Blood rheology improving agents

ABSTRACT

A blood rheology-improving agent comprising an antibody against human tissue factor (human TF).

TECHNICAL FIELD

[0001] The present invention relates to a blood rheology-improvingagent.

BACKGROUND ART

[0002] At the onset of pulmonary thrombosis or deep venous thrombosis,which are on the increase in recent years, thrombus formation due toreduced blood flow at rest or at recumbency is believed to be a leadingcause. Thus, in order to prevent or treat these diseases, there has beena demand for blood fluidity-improving agents i.e. bloodrheology-improving agents that prevent reduction in blood flow. However,no drugs are known that have an activity of effectively improving bloodrheology.

[0003] Thus, there has been a need for the development of drugs thatimprove blood rheology (fluidity).

DISCLOSURE OF THE INVENTION

[0004] The present invention provides a novel blood rheology-improvingagent.

[0005] After intensive and extensive study to solve the above problems,the present inventors have found that an antibody against human tissuefactor (sometimes referred to as anti-human TF antibody, or anti-TFantibody) can improve blood rheology.

[0006] Thus, the present invention provides a blood rheology-improvingagent comprising an anti-human TF antibody.

[0007] The above anti-human TF antibody can be a polyclonal antibody ora monoclonal antibody, or an altered antibody or an antibodymodification. Monoclonal antibodies can be generally produced byhybridomas and can also be produced by gene recombinant technology, andaltered antibodies and antibody modifications may generally be producedby gene recombinant technology. As altered antibodies, there can bementioned chimeric antibodies, for example human-mouse chimericantibodies, and as humanized antibodies, there can be mentioned versionsb-b, i-b, and i-b2 specifically described below. As antibodymodifications, there can be mentioned antibody fragments such as Fab,F(ab′)₂, and Fv, and single chain Fv (referred to as scFv) that has beenrendered single stranded by ligating the variable regions of antibody.

BRIEF EXPLANATION OF THE DRAWINGS

[0008]FIG. 1 is a graph that compares the antigen-binding activity of aH chain chimeric/L chain chimeric antibody, H chain humanized versionb/L chain humanized version b antibody, H chain humanized version i/Lchain humanized version b antibody, and H chain humanized version i/Lchain humanized version b2 antibody.

[0009]FIG. 2 is a graph that compares the neutralizing activity(activity of inhibiting Factor Xa production by TF) against human TF ofa H chain chimeric/L chain chimeric antibody, H chain humanized versionb/L chain humanized version b antibody, H chain humanized version i/Lchain humanized version b antibody, and H chain humanized version i/Lchain humanized version b2 antibody.

[0010]FIG. 3 is a graph that compares the neutralizing activity(activity of inhibiting Factor X binding) against human TF of a H chainchimeric/L chain chimeric antibody, H chain humanized version b/L chainhumanized version b antibody, H chain humanized version i/L chainhumanized version b antibody, and H chain humanized version i/L chainhumanized version b2 antibody.

[0011]FIG. 4 is a graph that compares the neutralizing activity(activity of inhibiting blood coagulation by TF) against human TF of a Hchain chimeric/L chain chimeric antibody, H chain humanized version b/Lchain humanized version b antibody, H chain humanized version i/L chainhumanized version b antibody, and H chain humanized version i/L chainhumanized version b2 antibody.

[0012]FIG. 5 is a graph showing that mononuclear cells treated withlipopolysaccharide (LPS) reduce blood fluidity.

[0013]FIG. 6 is a graph showing that blood fluidity that is reduced bylipopolysaccharide can be improved by anti-human tissue factor antibody.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0014] As used herein, the improvement of blood rheology means that theflow of blood becomes well.

[0015] Antibodies for use in the present invention may be any ofpolyclonal antibody and monoclonal antibody as long as they can improvethe rheology of blood, and monoclonal antibodies are preferred. Therecan be also used chimeric antibodies, humanized antibodies, single chainFv etc. based on monoclonal antibody. Humanized antibodies are mostpreferred.

[0016] 1. Anti-Human TF Antibody

[0017] Anti-human TF antibodies for use in the present invention may beof any origin, any type (monoclonal, polyclonal), and any shape, as longas they have an effect of improving blood rheology.

[0018] Anti-human TF antibodies for use in the present invention can beobtained as polyclonal or monoclonal antibodies using a known method. Asanti-human TF antibodies for use in the present invention, monoclonalantibodies of, in particular, a mammalian origin, are preferred.Monoclonal antibodies of a mammalian origin include those produced by ahybridoma and those produced by a host which has been transformed withan expression vector containing genetically engineered antibody gene.These antibodies, via binding to human TF, inhibit human TF to induce ahypercoagulable state.

[0019] 2. Antibody-Producing Hybridoma

[0020] A monoclonal antibody-producing hybridoma can be basicallycreated using a known procedure as described below. Thus, human TF or aportion (fragment) thereof may be used as a sensitizing antigen and isimmunized by the conventional method of immunization. The immune cellsthus obtained are fused with known parent cells in the conventional cellfusion process, and then the monoclonal antibody-producing hybridomasare screened by the conventional screening method to prepare the desiredhybridoma.

[0021] Specifically, the monoclonal antibody may be obtained in thefollowing manner.

[0022] First, human TF used as the sensitizing antigen for antibodygeneration can be obtained by expressing the human TF gene/amino acidsequence disclosed in J. H. Morrissey et al., Cell, Vol. 50, p. 129-135(1987). Thus, after the gene sequence encoding human TF is inserted intoa known expression vector system and a suitable host cell istransformed, the human TF protein of interest is purified from the hostcell or the culture supernatant thereof. This method is described inReference Example 1 of this specification. Furthermore, human TF used asthe antigen may be extracted from a TF-containing biological sample suchas a human placenta according to a method described in Reference Example2 and purified for use.

[0023] Then, the purified human TF protein is used as the sensitizingantigen. Alternatively, a soluble TF in which the transmembrane regionat the C-terminal end of human TF has been removed may be generated by,for example, recombinant DNA technology and may be used as thesensitizing antigen.

[0024] Mammals to be immunized with the sensitizing antigen arepreferably, but are not limited to, those selected in consideration oftheir compatibility with the parent cell for use in cell fusion. Theygenerally include rodents such as mice, rats and hamsters, or rabbits,monkeys and the like.

[0025] Immunization of animals with a sensitizing antigen is carried outusing a known method. A general method, for example, involves theintraperitoneal or subcutaneous administration of a sensitizing antigento the mammal. Specifically, a sensitizing antigen which has beendiluted and suspended in an appropriate amount of phosphate bufferedsaline (PBS) or physiological saline etc. is mixed, as desired, with anappropriate amount of a common adjuvant, for example Freund's completeadjuvant. After being emulsified, it is preferably administered to amammal for several times every 4 to 21 days. Alternatively a suitablecarrier may be used at the time of immunization of the sensitizingantigen.

[0026] After immunization of the mammal and the confirmation of theincrease in the desired antibody levels in the serum, the immune cellsare taken out from the mammal and are subjected to cell fusion.Preferred immune cells include in particular the spleen cells.

[0027] The mammalian myeloma cells are used as the other parent cellswhich are subjected to cell fusion with the above-mentioned immunecells. The myeloma cells preferably include various known cell linessuch as P3(P3X63Ag8.653) (Kearney, J. F. et al., J. Immunol. (1979) 123:1548-1550), P3X63Ag8U.1 (Yelton, D. E. et al., Current Topics inMicrobiology and Immunology (1978) 81: 1-7), NS-1 (Kohler, G. andMilstein, C., Eur. J. Immunol. (1976) δ: 511-519), MPC-11 (Margulies, D.H. et al., Cell (1976) δ: 405-415), SP2/0 (Shulman, M. et al., Nature(1978) 276: 269-270), FO (de St. Groth, S. F. et al., J. Immunol.Methods (1980) 35: 1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978)148: 313-323), R210 (Galfre, G. et al., Nature (1979) 277: 131-133) andthe like.

[0028] Cell fusion between the above immune cells and the myeloma cellsmay be essentially conducted in accordance with a known method such asis described in Milstein et al. (Kohler, G. and Milstein, C., MethodsEnzymol. (1981) 73: 3-46).

[0029] More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and, in addition, an adjuvant such as dimethyl sulfoxide etc. may beadded as desired to enhance the efficiency of fusion.

[0030] The preferred ratio of the immune cells and the myeloma cells tobe used is, for example, 1 to 10 times more immune cells than themyeloma cells. Examples of culture media to be used for the above cellfusion include RPMI1640 medium and MEM culture medium suitable for thegrowth of the above myeloma cell lines, and the conventional culturemedium used for this type of cell culture and, besides, a serumsupplement such as fetal calf serum (FCS) may be added.

[0031] In cell fusion, predetermined amounts of the above immune cellsand the myeloma cells are mixed well in the above culture liquid, towhich a PEG solution previously heated to about 37° C., for example aPEG solution (a mean molecular weight of about 1000 to 6000), is addedat a concentration of 30 to 60%(w/v) and mixed to obtain the desiredfusion cells (hybridomas). Then, by repeating the sequential addition ofa suitable culture liquid and centrifugation to remove the supernatant,cell fusion agents etc. which are undesirable for the growth of thehybridoma, can be removed.

[0032] The hybridoma thus obtained is selected by culturing in theconventional selection medium, for example, the HAT culture medium (aculture liquid containing hypoxanthine, aminopterin, and thymidine).Culturing in said HAT culture medium is continued generally for a periodof time sufficient to effect killing of the cells (non-fusion cells)other than the desired hybridoma, generally several days to severalweeks. Then, the conventional limiting dilution method is conducted inwhich the hybridomas that produce the desired antibody are screened andmonoclonally cloned.

[0033] In addition to obtaining the above hybridoma by immunizing ananimal other than the human with an antigen, it is also possible tosensitize human lymphocytes in vitro with human TF, and the resultingsensitized B lymphocytes are fused with a human myeloma cell capable ofdividing permanently to obtain the desired human antibody having theactivity of binding to human TF (see Japanese Post-examined PatentPublication (Kokoku) No. 1-59878). Furthermore, a transgenic animalhaving a repertoire of all or some human antibody genes is immunizedwith the antigen human TF to obtain human TF antibody-producing cells,which are then immortalized, from which the desired human antibodyagainst human TF may be obtained (see International Patent PublicationWO 94/25585, wo 93/12227, Wo 92/03918, wO 94/02602, WO 96/34096, and WO96/33735).

[0034] The monoclonal antibody-producing hybridomas thus constructed canbe subcultured in the conventional culture liquid, or can be stored fora prolonged period of time in liquid nitrogen.

[0035] In order to obtain monoclonal antibodies from said hybridoma, amethod can be used in which said hybridoma is cultured in theconventional method and the antibodies are obtained as the supernatant,or a method in which the hybridoma is administered to and grown in amammal compatible with said hybridoma and the antibodies are obtained asthe ascites. The former method is suitable for obtaining high-purityantibodies, whereas the latter is suitable for a large scale productionof antibodies.

[0036] An example of monoclonal antibody production is specificallydescribed in Reference Example 2. In this example, six monoclonalantibodies (termed as ATR-2, 3, 4, 5, 7, and 8) have been obtained. Allof them can be used in the present invention, but ATR-5 is mostpreferred.

[0037] 3. Recombinant Antibody

[0038] A recombinant antibody, which was produced by the recombinantgene technology in which an antibody gene was cloned from the hybridomaand integrated into a suitable vector which was then introduced into ahost, can be used in the present invention as monoclonal antibody (see,for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192,767-775).

[0039] Specifically, mRNA encoding the variable (V) region of anti-humanTF antibody is isolated from the anti-human TF antibody-producinghybridoma. The isolation of mRNA is conducted by preparing total RNAusing, for example, a known method such as the guanidine ultracentrifugemethod (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), theAGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162,156-159), and then mRNA is purified from the total RNA using an mRNApurification kit (manufactured by Pharmacia) and the like.Alternatively, mRNA can be directly prepared using the Quick Prep mRNAPurification Kit (manufactured by Pharmacia).

[0040] cDNA of the V region of antibody may be synthesized from the mRNAthus obtained using a reverse transcriptase. cDNA may be synthesizedusing the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(manufactured by Seikagaku Kogyo) and the like. Alternatively, for thesynthesis and amplification of cDNA, the 5′-Ampli FINDER RACE Kit(manufactured by Clontech) and the 5′-RACE method (Frohman, M. A. etal., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. etal., Nucleic Acids Res. (1989) 17, 2919-2932) that employs polymerasechain reaction (PCR) may be used.

[0041] The desired DNA fragment is purified from the PCR productobtained and may be ligated to vector DNA. Moreover, a recombinantvector is constructed therefrom, and then is introduced into E. colietc., from which colonies are selected to prepare the desiredrecombinant vector. The base sequence of the desired DNA may beconfirmed by a known method such as the dideoxy nucleotide chaintermination method.

[0042] Once DNA encoding the V region of the desired anti-human TFantibody has been obtained, it may be integrated into an expressionvector containing DNA encoding the constant region (C region) of thedesired antibody.

[0043] In order to produce anti-human TF antibody for use in the presentinvention, the antibody gene is integrated as described below into anexpression vector so as to be expressed under the control of anexpression regulatory region, for example an enhancer and/or a promoter.Subsequently, a host cell may be transformed by the expression vectorand the antibody can then be expressed therein.

[0044] For expression of the antibody gene, DNA encoding the heavy chain(H chain) or the light chain (L chain) of antibody may be separatelyintegrated into expression vectors and the hosts are transformedsimultaneously, or DNA encoding the H chain and the L chain may beintegrated into a single expression vector and the host is transformedtherewith (see International Patent Publication WO 94-11523).

[0045] Furthermore, in addition to the above host cells, transgenicanimals may be used for the production of recombinant antibody. Forexample, an antibody gene is inserted into the middle of the geneencoding protein (such as goat β casein) which is inherently produced inthe milk to prepare fusion genes. DNA fragments containing the fusiongene into which the antibody gene has been inserted are injected into agoat embryo, and the embryo is introduced into a female goat. Thedesired antibody is obtained from the milk produced by the transgenicgoat born to the goat who received the embryo or offsprings thereof. Inorder to increase the amount of milk containing the desired antibodyproduced by the transgenic goat, hormones may be given to the transgenicgoat as appropriate. (Ebert, K. M. et al., Bio/Technology (1994) 12,699-702).

[0046] An example of the method of producing recombinant antibody isspecifically described in Reference Example 3.

[0047] 4. Altered Antibody

[0048] In accordance with the present invention, artificially alteredrecombinant antibody such as chimeric antibody and humanized antibodycan be used for the purpose of lowering heterologous antigenicityagainst humans. These altered antibody can be produced using knownmethods.

[0049] Chimeric antibody can be obtained by ligating the thus obtainedDNA encoding the V region of antibody to DNA encoding the C region ofhuman antibody, which is then integrated into an expression vector andintroduced into a host for the production of the antibody therein. Usingthis known method, chimeric antibody useful for the present inventioncan be obtained.

[0050] Humanized antibody which is also called reshaped human antibodyhas been generated by transplanting the complementarity determiningregion (CDR) of antibody of a mammal other than the human, for examplemouse antibody, into the CDR of human antibody. A general recombinantDNA technology for preparation of such antibodies is also known (seeEuropean Patent Application EP 125023 and WO 96/02576).

[0051] Specifically, a DNA sequence which was designed to ligate the CDRof mouse antibody with the framework region (FR) of human antibody issynthesized by the PCR method using, as the primers, several dividedoligonucleotides that were constructed so as to have sectionsoverlapping with one another at the ends of the CDR and the FR (see themethod described in International Patent Publication WO 98/13388)

[0052] For the framework region of human antibody ligated through CDR, acomplementarity determining region that forms a favorable antigenbinding site is selected. When desired, amino acids in the frameworkregion of the antibody variable region may be substituted so that thecomplementarity determining region of reshaped human antibody may forman appropriate antigen biding site (Sato, K. et al., Cancer Res. (1993)53, 851-856).

[0053] For example, for chimeric antibody or humanized antibody, the Cregion of human antibody is used. For H chain, for example, Cγ1, Cγ2,Cγ3, and Cγ4 can be used, and for L chain, CK and Ck can be used. The Cregion of human antibody may be modified to improve the stability ofantibody or the production thereof.

[0054] Chimeric antibody consists of the variable region of antibodyderived from a mammal other than the human and the constant regionderived from human antibody, whereas humanized antibody consists of thecomplementarity determining region of antibody derived from a mammalother than the human and the framework region and the C region derivedfrom human antibody. Accordingly, antigenicity thereof in the human bodyhas been reduced so that they are useful as an active ingredient of atherapeutic agent for use in the present invention.

[0055] The method of constructing chimeric antibody is specificallydescribed in Reference Example 4.

[0056] The method of constructing humanized antibody is specificallydescribed in Reference Example 5.

[0057] In this Reference Example, as the humanized heavy chain (H chain)variable region (v region), versions a, b, c, d, e, f, g, h, i, j, b1,d1, b3 and d3 having the amino acid sequence shown in Table 1 and Table2 were used. TABLE 1 The amino acid sequence of H chain V region FR1CDR1 FR2 CDR2          1         2         3     4 5          6123456789012345678901234567890 12345 67890123456789 012A3456789012345L39130(a) QVQLLESGAVLARPGTSVKISCKASGFNIK DYYMH WVKQRPGQGLEWIGGNDPANGHSMYDPKFQG Z34963(b) ------------------------------ ------------------- ----------------- M30885(c)------------------------------ ----- -------------- -----------------M62723(d) ------------------------------ ----- ------------------------------- Z80844(e) ------------------------------ ------------------- ----------------- L04345(f)------------------------------ ----- -------------- -----------------S78322(g) ------------------------------ ----- ------------------------------- Z26827(h) ------------------------------ ------------------- ----------------- U95239(i)------------------------------ ----- -------------- -----------------L03147(j) ------------------------------ ----- ------------------------------- P01742(b1) ------------------------------ -------R-A-------M- ----------------- P01742(d1)------------------------------ ----- --R-A-------M- -----------------Z80844(b3) ------------------------------ ----- --R-A-------------------------- Z80844(d3) ------------------------------ -------R-A--------- -----------------

[0058] TABLE 2 The amino acid sequence of H chain V reqion (continuedfrom Table 1) FR3 CDR3 FR4     7         8            9     10       1167890123456789012ABC345678901234 56789012 34567890123 L39130(a)RAKLTAATSASIAYLEFSSLTNEDSAVYYCAR DSGYAMDY WGQGTLVTVSS Z34963(b)-VTI--D--TNT--M-L---RS--T-I----- -------- ----------- M30885(c)-VTMLVD--KNQFS-RL--V-AA-T------- -------- ----------- M62723(d)-VTI--DE-T-T--M-L---RS------F--- -------- ----------- Z80844(e)-VSI--DE-TK---M-LN--RS--T---F--- -------- ----------- L04345(f)-VTI--DT-T-T--M-LR--RSD-T------- -------- ----------- S78322(g)K-T---DE-S-T--MQL---RS------S--- -------- ----------- Z26827(h)-VTMS-DK-S-A---QWT--KAS-T-I-F--- -------- ----------- U95239(i)-VTI--D--T-TVFM-L---RS--T------- -------- ----------- L03147(j)-VTF--D---NT--M-LR--RSA-T------- -------- ----------- P01742(b1)-VTI--D--TNT--M-L---RS--T-I----- -------- ----------- P01742(d1)-VTI--DE-T-T--M-L---RS------F---- -------- ----------- Z80844(b3)-VTI--D--TNT--M-L---RS--T-I----- -------- ----------- Z80844(d3)-VTI--DE-T-T--M-L---RS------F--- -------- -----------

[0059] Also, as the humanized light chain (L chain) V region, versionsa, b, c, b1 and b2 having the amino acid sequence shown in Table 3 wereused. TABLE 3 The amino acid sequence of L chain V region FR1 CDR1 FR2CDR2          1         2       3      4 5 1234567890123456789012345678901234 567890123456789 0123456 Z37332(a) DIQMTQSPSSLSASVGDRVTITCKASQDIKSFLS WYQQKPGKAPKLLIY 40 YATSLAD S68699(b) ---------------------------------- --------------- ------- P01607(c) ---------------------------------- --------------- ------- S65921(b1) ---------------------------------- -F------S--T--- ------- X93625(b2) ---------------------------------- ------E----S--- ------- FR3 CDR3 FR4   6         7         8  9  10 78901234567890123456789012345678901234567 8901234567 Z37332(a) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHGESPYT FGGGTKVEIK S68699(b) --------------Y-------------------------- ---------- P01607(c) --------------Y-----------I-------------- ---------- S65921(b1) --------------Y-------------------------- ---------- X93625(b2) --------------Y-------------------------- ----------

[0060] Various versions of the above H chain V region and variousversions of the L chain V region were combined and their antigen bindingability and the TF-neutralizing activity were evaluated. As shown inReference Example 6 and Reference Example 7, it was shown that whenexpressed as “H chain V region version”—“L chain V region version”,“b-b”, “i-b” and “i-b2” exhibited particularly high activities. Theantigen-binding ability of these humanized antibodies is shown in FIG.1, the activity of neutralizing human TF (the activity of inhibiting theproduction of Factor Xa by TF) is shown in FIG. 2, the activity ofneutralizing human TF (the activity of inhibiting the production ofFactor X by TF) is shown in FIG. 3, and the activity of neutralizinghuman TF (the activity of inhibiting plasma coagulation by TF) is shownin FIG. 4.

[0061] 5. Antibody Modifications

[0062] Antibodies for use in the present invention may be antibodyfragments or modified versions thereof as long as they bind to human TFand inhibit the activity of human TF. For example, as fragments ofantibody, there may be mentioned Fab, F(ab′)₂, Fv or single-chain Fv(scFv) in which Fv's of H chain or L chain were ligated via a suitablelinker.

[0063] Specifically, antibodies are treated with an enzyme, for example,papain or pepsin, to produce antibody fragments, or genes encoding theseantibody fragments are constructed, and then introduced into anexpression vector, which is expressed in a suitable host cell (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M.and Horwitz, A. H., Methods in Enzymology (1989) 178, 476-496;Plucktrun, A. and Skerra, A., Methods in Enzymology (1989) 178, 497-515;Lamoyi, E., Methods in Enzymology (1986) 121, 652-663; Rousseaux, J. etal., Methods in Enzymology (1986) 121, 663-669; Bird, R. E. et al.,TIBTECH (1991) 9, 132-137).

[0064] scFv can be obtained by ligating the V region of H chain and theV region of L chain of antibody. In the scFv, the V region of H chainand the V region of L chain are ligated via a linker, preferably apeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988)85, 5879-5883). The V region of H chain and the V region of L chain inthe scFv may be derived from any of the above-mentioned antibodies. Asthe peptide linker for ligating the V regions, any single-chain peptidecomprising, for example, 12-19 amino acid residues may be used.

[0065] DNA encoding scFv can be obtained using DNA encoding the H chainor the H chain V region of the above antibody and DNA encoding the Lchain or the L chain V region of the above antibody as the template byamplifying the whole or the portion of the DNA encoding the desiredamino acid sequence among the above sequences by the PCR technique withthe primer pair specifying the both ends thereof, and by furtheramplifying the combination of DNA encoding the peptide linker portionand the primer pair which defines that both ends of said DNA be ligatedto the H chain and the L chain, respectively.

[0066] Once DNA encoding scFv is constructed, an expression vectorcontaining it and a host transformed with said expression vector can beobtained by conventional methods, and scFv can be obtained using theresultant host by the conventional methods.

[0067] These antibody fragments can be produced by obtaining the genethereof in a similar manner to that mentioned above and by allowing itto be expressed in a host. “Antibody” as used herein encompasses theseantibody fragments.

[0068] As antibody modifications, anti-human TF antibody associated withvarious molecules such as polyethylene glycol (PEG) can be used.“Antibody” as used herein encompasses these antibody modifications.These antibody modifications can be obtained by chemically modifying theantibodies thus obtained. These methods have already been established inthe art.

[0069] 6. Expression and Production of Recombinant Antibody or AlteredAntibody

[0070] The antibody gene constructed as described above can be expressedand obtained using a known method. In the case of a mammal, a commonlyused useful promoter, the human tissue factor gene, and a poly A signalto 3′-end downstream thereof can be operably linked and can beexpressed. As the promoter/enhancer, for example, there can be mentionedhuman cytomegalovirus immediate early promoter/enhancer.

[0071] As other promoters/enhancers that may be used for the expressionof antibody for use in the present invention, there can be mentionedviral promoters/enhancers of retrovirus, polyoma virus, adenovirus,simian virus 40 (SV40), and the like, and promoters/enhancers derivedfrom mammalian cells such as human elongation factor 1α (HEF1α).

[0072] For example, gene expression may be readily accomplished by themethod of Mulligan et al. (Nature (1979) 277, 108-114) when Sv40promoter/enhancer is used, or by the method of Mizushima et al. (NucleicAcids Res. (1990) 18, 5322) when HEF1α promoter/enhancer is used.

[0073] In the case of E. coli, expression may be conducted by operablylinking a commonly used useful promoter, a signal sequence for antibodysecretion, and the antibody gene to be expressed, followed by expressionthereof. As the promoter, for example, there can be mentioned laczpromoter and araB promoter. The method of Ward et al. (Nature (1098)341, 544-546; Ward, E. S. et al., FASEB J. (1992) 6, 2422-2427) may beused when lacz promoter is used, and the method of Better et al.(Science (1988) 240, 1041-1043) may be used when araB promoter is used.

[0074] As the signal sequence for antibody secretion, when produced inthe periplasm of E. coli, the pelB signal sequence (Lei, S. P. et al.,J. Bacteriol. (1987) 169, 4379-4383) can be used. After separating theantibody produced in the periplasm, the structure of the antibody isappropriately refolded before use.

[0075] As the origin of replication, there can be used those derivedfrom SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) andthe like. Furthermore, for the amplification of the gene copy number inthe host cell system, expression vectors can include as selectablemarkers the aminoglycoside phosphotransferase (APH) gene, the thymidinekinase (TK) gene, E. coli xanthine guaninephosphoribosyl transferase(Ecogpt) gene, the dihydrofolate reductase (dhfr) gene and the like.

[0076] For the production of antibody for use in the present invention,any production system can be used, for example a production system whichemploys eukaryotic cells and the production system which employsprokaryotic cells. As the eukaryotic cells, there can be mentioned, forexample, the mammalian cell system, the insect cell system, and thefungal cell system such as filamentous fungal cells and yeast cells, andas the prokaryotic cells, there can be mentioned, for example, bacterialcells such as E. coli cells.

[0077] Preferably the antibody for use in the present invention may beexpressed in mammalian cells such as CHO cells, COS cells, myelomacells, baby hamster kidney (BHK) cells, Vero cells, and HeLa cells.

[0078] By culturing the transformed cells in vivo or in vitro, theantibody of interest can be obtained. Culturing is conducted by a knownmethod. For example, as the culture liquid, DMEM, MEM, RPMI1640, andIMDM can be used, and serum supplements such as fetal calf serum (FCS)may be used in combination.

[0079] 7. Separation and Purification of Antibody

[0080] Antibodies produced and expressed as described above can beseparated from the host cell and then may be purified to homogeneity.Separation and purification of the antibody for use in the presentinvention may be accomplished by affinity chromatography. As the columnthat employs Protein A column, there can be mentioned Hyper D, POROS,Sepharose F. F. (manufactured by Pharmacia) and the like. Alternatively,methods for separation and purification conventionally used for proteinscan be used without any limitation. Separation and purification of theantibody may be accomplished by combining, as appropriate,chromatography other than the above-mentioned affinity chromatography,filtration, ultrafiltration, salting-out, dialysis and the like.(Antibodies: A Laboratory Manual. Ed. Harlow and David Lane, Cold SpringHarbor Laboratory, 1988).

[0081] 8. Confirmation of the Effect of Improving Blood Rheology

[0082] Blood rheology can be observed by determining the blood flow ofperipheral blood in vitro using a cell micro rheology measurementinstrument, etc. Treatment with LPS caused reduced blood flow, but theblood flow was improved by the addition of the anti-human TF antibody ofthe present invention. It was confirmed therefore that the anti-human TFantibody of the present invention is effective for improving bloodrheology. Diseases that require such improvement includearteriosclerosis, pulmonary embolism, deep venous thrombosis, chronicarterial obstruction, etc., and the reduced blood flow associated withthese diseases can be improved by anti-human TF antibody of the presentinvention.

[0083] The effect is specifically described in the Examples.

[0084] 9. Method of Administration and Formulation

[0085] The therapeutic agent of the present invention is used for thepurpose of improving blood rheology.

[0086] Effective dosage per administration is selected from the range of0.001 mg to 1000 mg/kg body weight. Alternatively, the dosage of 0.01 to100 mg/kg, preferably 0.1 to 10 mg/kg may be selected. However, thetherapeutic agent containing anti-human TF antibody of the presentinvention is not limited to these dosages.

[0087] Preferably the method of administration is, but is not limitedto, intravenous injection, intravenous drip, and the like.

[0088] The therapeutic agent of the present invention comprisinganti-human TF antibody as an active ingredient may be formulated using astandard method (Remington's Pharmaceutical Science, the latest edition,Mark Publishing Company, Easton, USA), and may contain pharmaceuticallyacceptable carriers and/or additives.

[0089] Examples of such carriers or additives include water, apharmaceutically acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer,carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate,water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, agar,polyethylene glycol, diglycerin, glycerin, propylene glycol, Vaseline,paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA),mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactantand the like.

[0090] Additives used are chosen from, but not limited to, the above orcombinations thereof, as appropriate, depending on the dosage form ofthe present invention. For example, when used as injections, purifiedanti-human TF antibody may be dissolved in a solvent such asphysiological saline, a buffer, and a glucose solution, to which ananti-adsorbent such as Tween 80, Tween 20, gelatin, and human serumalbumin may be added. Alternatively, they may be lyophilized so as to bedissolved and reconstituted into a dosage form before use. As theexcipient for lyophilization, sugar alcohols and sugars such as mannitoland glucose may be used.

EXAMPLES

[0091] The present invention will now be explained more specificallywith reference to Examples.

[0092] Three-point-five ml of peripheral blood is added to 3 ml ofMono-Poly separation solution, and is centrifuged at 1,500 rpm (×700G)for 20 minutes. The mononuclear cell fraction thus collected is washedwith phosphate buffered saline (PBS) in a 50 ml Falcon tube, andcentrifuged to collect cell pellets, which are dispersed at variousconcentrations in a 10% PBS-containing RPMI 1640 medium containinglipopolysaccharide (LPS). The concentration of lipopolysaccharide wasthe 0 μg/ml no-addition group (negative control) and the 4.0 μg/mladdition group.

[0093] The cell suspension thus prepared was incubated in a 100 mmculture dish in a CO₂ incubator for 6 hours. Then, the cells attached tothe culture dish were scraped by a scraper, and after adjusting the cellconcentration to 6×10⁵ in phosphate buffered saline (PBS), they werecentrifuged at 800 rpm (×200G) for 10 minutes, and the cell pellets werecollected, washed in phosphate buffered saline (PBS), and centrifuged tocollect cell pellets.

[0094] Then, in order to investigate blood coagulation activity of theabove lipopolysaccharide-treated mononuclear cells, 6×10⁵ cells wereadded to 1000 μl of whole blood. After the mixture was incubated at roomtemperature for 10 minutes, 500 ng of activated Factor 7 (FVIIa) andCaCl₂ at a final concentration of 5 mM were added thereto, which wasapplied to a cell micro rheology measurement instrument (MCFAN-KH;Hitachi Haramachi Electronics Co., Ltd). This instrument simulates thecapillary blood vessels, and is constructed in such a way that amultitude of fine V-shaped grooves are formed on silicon single crystal,whereupon a glass substrate has been pressure-deposited to formartificial capillary blood vessels, and through the glass substrate, theflow of blood cells can be observed using a microscope. The result isexpressed in the volume of blood that flowed through the capillary bloodvessels per unit time and is shown in FIG. 5. From the figure, it wasconfirmed that the fluidity of the blood decreases depending on theamount of polyliposaccharide.

[0095] Thus, at the time of incubation of mixture of the abovelipopolysaccharide-treated (4 μg/ml) mononuclear cells and whole blood,anti-human tissue factor antibody ATR-5 (produced in Reference Example2) was added at 1.25 μg/ml, 2.5 μg/ml, or 5 μg/ml, or not added(negative control), and incubated. As the positive control, a test wascarried out by adding mononuclear cells not treated withlipopolysaccharide to whole blood. Then, the fluidity of the incubatedmixture was measured as described above using the cell micro rheologymeasurement instrument. The result is shown in FIG. 6. It was confirmedthat anti-human tissue factor antibody improves blood fluidity.

Reference Example 1 Method of Preparing Soluble Human TF

[0096] Soluble human TF (shTF) was prepared in the following manner.

[0097] The gene encoding the human TF penetrating region (amino acid atposition 220) and thereafter that had been replaced with the FLAGpeptide M2 was inserted to a mammalian cell expression vector(containing the neomycin resistant gene and the DHFR gene), andintroduced into CHO cells. For the cDNA sequence of human TF, referencewas made to an article by James H. Morrissey et al. (Cell (1987) 50:129-135). The gene sequence and the amino acid sequence of this solublehuman TF are shown in SEQ ID NO: 101 and 102. After drug selection withG418, the expressing cells were selected, which were then subjected toexpression amplification with methotrexate, and the shTF-expressingcells were established.

[0098] The cells were cultured in the serum-free medium CHO-S-SFMII(GIBCO) to obtain a culture supernatant containing shTF. The supernatantwas diluted two times with an equal volume of a 40 mM Tris-HCl buffer(pH 8.5), which was added to the Q-Sepharose Fast Flow column (100 ml,Pharmacia Biotech) equilibrated with a 20 mM Tris-HCl buffer (pH 8.5).After washing with the same buffer containing 0.1 M NaCl, theconcentration of NaCl was changed to 0.3 M, and shTF was eluted from thecolumn. To the shTF fraction obtained, ammonium sulfate was added to afinal concentration of 2.5 M, and was centrifuged (10,000 rpm, 20minutes) to precipitate the contaminating proteins. The supernatant wasadded to Butyl TOYOPEARL (30 ml, TOSOH), and then was washed with a 50mM Tris-HCl buffer (pH 6.8) containing 2.5 M ammonium sulfate.

[0099] In the 50 mM Tris-HCl buffer (pH 6.8), the concentration ofammonium sulfate was linearly reduced from 2.5 M to 0 M to permit theelution of shTF. The peak fractions containing shTF were concentrated bythe Centri-Prep 10 (Amicon). The concentrate was added to the TSKgelG3000SWG column (21.5×600 mm, TOSOH) equilibrated with a 20 mM Tris-HClbuffer (pH 7.0) containing 150 mM NaCl, and the peak fraction of shTFwas collected. It was filter sterilized with a 0.22 μm membrane filterand the product was set as the soluble human TF (shTF). Theconcentration of the sample was calculated assuming that the molarextinction coefficient of the sample ε=40,130 and molecularweight=43,210.

Reference Example 2 Preparation of Anti-TF Monoclonal Antibody

[0100] 1. Purification of Human TF

[0101] The purification of TF from human placenta was carried outaccording to the method of Ito (Ito, T. et al., J. Biol. Chem., 114:691-696, 1993). Thus, human placenta was homogenized in a Tris bufferedsaline (TBS, pH 7.5) containing 10 mM benzamidine hydrochloride, 1 mMphenylmethylsulfonyl fluoride, 1 mM diisopropylfluoro phosphate, and0.02% sodium azide, and then the precipitate was defatted with coldacetone. The defatted powder obtained was suspended in the above buffercontaining 2% Triton X-100 to solubilize TF.

[0102] The supernatant was subjected to affinity chromatography usingConcanavalin A-Sepharose 4B column (Pharmacia) and anti-TFantibody-bound Sepharose 4B column (Pharmacia) to obtain purified TF.This was concentrated with an ultrafiltration membrane (PM-10, Amicon)and was stored, as the purified sample, at 4° C.

[0103] TF content in the purified sample was quantitated by SandwichELISA that combined a commercially available anti-TF monoclonal antibody(American Diagnostica) and polyclonal antibody (American Diagnostica)with recombinant TF as a standard.

[0104] The purity in the purified sample was confirmed by subjecting thesample to SDS-PAGE using a 4-20% density gradient polyacrylamide gel,and silver-staining the product.

[0105] 2. Immunization and the Preparation of Hybridoma

[0106] After mixing the purified human TF (about 70 μg/ml) with an equalvolume of Freund's complete adjuvant (Difco), it was immunizedsubcutaneously into the abdomen of 5-week old Balb/c male mice (NipponCharles River) at 10 μg TF/mouse. On day 12, 18, and 25 after theinitial immunization, TF mixed with Freund's incomplete adjuvant wassubcutaneously boosted at 5 μg/mouse TF and, as a final immunization,the TF solution diluted with PBS was intraperitoneally given at 5μg/mouse on day 32.

[0107] Three days after the final immunization, the spleen cells wereprepared from four mice, and were fused to the mouse myeloma cell lineP3U1 at about ⅕ cell count thereof by the polyethylene glycol method.The fused cells were suspended into the RPMI-1640 medium

[0108] (hereinafter referred to as RPMI-medium)

[0109] (Lifetechoriental) containing 10% fetal bovine serum, which wasinoculated in 400 wells per mouse of a 96-well plate. On day 1, 2, 3,and 5 after the fusion, half the volume of the medium was exchanged withthe RPMI-medium (hereinafter referred to as HAT-medium) containing HAT(Dainippon Seiyaku) and condimed H1 (Boehringer Mannheim GmbH) toperform HAT selection of the hybridoma.

[0110] The hybridomas selected by the screening method described belowwere cloned by conducting limiting dilution twice.

[0111] For the limiting dilution, 0.8 cells was inoculated per well intwo 96-well plates. For the wells in which a single colony was confirmedby microscopic examination, clones were selected by the followingmeasurement of the TF-binding activity and TF-neutralizing activity. Theclones obtained were acclimated from the HAT-medium to the RPMI-medium.After confirming the absence of reduction in antibody production abilitydue to acclimation, limiting dilution was performed again for completecloning. By the foregoing procedure, hybridomas that produce sixantibodies (ATR-2, 3, 4, 5, 7, and 8) that strongly inhibit the bindingof TF/Factor VIIa complex and Factor X were established.

[0112] 3. Ascites Formation and Antibody Purification

[0113] The ascites formation of the established hybridomas were carriedout according to the standard method. Thus, 106 hybridomas that weresubcultured in vitro were intraperitoneally grafted into BALB/C malemice that had previously received two intravenous administrations ofmineral oil. Ascites was collected from the mice that showed a bloatedabdomen 1-2 weeks after the grafting.

[0114] The purification of antibody from ascites was carried out usingthe ConSepLC100 system (Millipore) equipped with the Protein A column(Nippon Gaishi).

[0115] 4. Cell-ELISA

[0116] Human cystocarcinoma cell line J82 (Fair D. S. et al., J. Biol.Chem., 262: 11692-11698, 1987) that is known to express TF at a highlevel was obtained from ATCC, and subcultured and maintained in theRPMI-medium under the condition of 37° C., 5% CO₂, and 100% humidity.

[0117] Cell-ELISA plates were prepared by inoculating J82 cells to a96-well plate at 10⁵ cells/well, culturing for one day under the abovecondition, removing the medium and then washing twice with phosphatebuffered saline (PBS), adding a 4% paraformaldehyde solution (PFA), andallowing to stand on ice, for 10 minutes, for immobilization. After PFAwas removed, the plate was washed with PBS, the Tris buffer (Blockingbuffer) containing 1% BSA and 0.02% sodium azide was added thereto, andthe plate was stored at 4° C. until use.

[0118] Cell-ELISA was carried out in the following manner. Thus, theBlocking buffer was removed from the plate prepared as above, to whichan anti-TF antibody solution or a hybridoma culture supernatant wasadded and was reacted at room temperature for 1.5 hours. After washingwith PBS containing 0.05% Tween 20, alkaline phosphatase-conjugated goatanti-mouse IgG (H+L) (Zymed) was reacted for 1 hour. After washing, 1mg/ml p-nitrophenyl phosphate disodium (Sigma) was added, and one hourlater absorbance at 405 nm was measured to determine the amount ofanti-TF antibody that bound to the J82 cells.

[0119] 5. Assay System of TF-Neutralizing Activity with Factor XaActivity as an Index

[0120] To 50 μl of Tris buffered saline (TBS: pH 7.6) containing 5 mMCaCl₂ and 0.1% bovine serum albumin, 10 μl of a human placenta-derivedthromboplastin solution (5 mg/ml) (Thromborel S) (Boehring) and 10 μl ofa Factor VIIa solution (82.5 ng/ml) (American Diagnostica) were added,and reacted at room temperature for 1 hour to permit the formation ofthe TF/Factor VIIa complex. After 10 μl of a predetermined concentrationof a diluted anti-TF antibody solution or the hybridoma culturesupernatant and 10 μl of a Factor X solution (3.245 μg/ml) (CelsusLaboratories) were added and reacted for 45 minutes, 10 μl of 0.5 M EDTAwas added to stop the reaction. Fifty μl of 2 mM S-2222 solution(Daiichi Kagaku Yakuhin) was added thereto, and changes in absorbance at405 nm over 30 minutes were measured and were set as the FactorX-producing activity of TF. In this method, the activity of antibodythat inhibits the binding of the TF/Factor VIIa complex and Factor X canbe determined.

[0121] 6. Assay System of Plasma Coagulation-Inhibiting Activity

[0122] Fifty μl of an appropriately diluted anti-TF antibody solutionwas mixed with 100 μl of a commercially available normal human plasma(Kojin Bio) and reacted at 37° C. for 3 minutes. Then 50 μl of a humanplacenta-derived thromboplastin solution (1.25 mg/ml) was added thereto,and the time to coagulation of the plasma was measured using the plasmacoagulation measuring instrument (CR-A: Amelung).

[0123] 7. Determination of Antibody Isotype

[0124] For the culture supernatant of the hybridoma and the purifiedantibody, the mouse monoclonal antibody isotyping kit (manufactured byAmersham) was used to confirm the isotype of antibody. The result isshown below. TABLE 5 Immunoglobulin isotype of anti-TF monoclonalantibody ATR-2 IgG1, k ATR-3 IgG1, k ATR-4 IgG1, k ATR-5 IgG1, k ATR-7IgG2a, k ATR-8 IgG2a, k

Reference Example 3 Cloning of DNA Encoding the V Region of a MouseMonoclonal Antibody Against Human TF

[0125] (1) Preparation of mRNA

[0126] mRNA was prepared from hybridoma obtained in Reference Example 2ATR-5 using the QuickPrep mRNA Purification Kit (Pharmacia Biotech).Each hybridoma cell was completely homogenized in the extraction bufferaccording to instructions attached to the kit, and then mRNA waspurified by the oligo (dT)-cellulose spun column, followed by ethanolprecipitation. The mRNA precipitate was dissolved in the elution buffer.

[0127] (2) Preparation and Amplification of cDNA of the Gene Encoding aMouse Antibody V Region

[0128] (i) Cloning of H Chain V Region cDNA

[0129] The cloning of the gene encoding the H chain V region of a mousemonoclonal antibody against human TF was carried out using the 5′-RACEmethod (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002,1988; Belyavsky, A. et al., Nucleic Acids Res. 17: 2919-2932, 1989). Forthe 5′-RACE method, the Marathon cDNA Amplification Kit (CLONTECH) wasused and the procedure carried out according to the instructionsattached to the kit.

[0130] Using about 1 μg of mRNA prepared as above as a template, thecDNA synthesis primer attached to the kit was added, which was reactedwith a reverse transcriptase at 42° C. for 60 minutes to effect reversetranscription to cDNA. This was reacted with DNA polymerase I, DNAligase, and RNaseH at 16° C. for 1.5 hour, and with T4 DNA polymerase at16° C. for 45 minutes thereby to synthesize a double stranded cDNA. Thedouble stranded cDNA was extracted with phenol and chloroform, andrecovered by ethanol precipitation.

[0131] By overnight reaction with T4 DNA ligase at 16° C., a cDNAadapter was ligated to both ends of the double stranded cDNA. Thereaction mixture was diluted 50-fold with a 10 mM Tricine-KOH (pH 8.5)containing 0.1 mM EDTA. Using this as a template, the gene encoding theH chain V region was amplified by PCR. The adapter primer 1 attached tothe kit was used for the 5′-end primer, and for the 3′-end primer theMHC-G1 primer (SEQ ID NO: 1) (S. T. Jones, et al., Biotechnology, 9:88-89, 1991) were used.

[0132] PCR solutions for the ATR-5 antibody H chain V region contained,in 100 μl, 120 mM Tris-HCl (pH 8.0), 10 mM KCl, 6 mM (NH₄)₂SO₄, 0.1%Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 mMMgCl₂, 2.5 units of KOD DNA polymerase (Toyo Boseki), 30-50 pmole ofadapter primer 1, as well as MHC-G1 primer, and 1-5 μl of a reactionmixture of cDNA to which the cDNA adapter was ligated.

[0133] All PCRs were carried out using the DNA Thermal Cycler 480(Perkin-Elmer), and the PCR was performed for thirty cycles at atemperature cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and74° C. for 1 minute.

[0134] (ii) Cloning of L Chain V Region cDNA

[0135] The cloning of the gene encoding the L chain V region of a mousemonoclonal antibody against human TF was carried out using the 5′-RACEmethod (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002,1988; Belyavsky, A. et al., Nucleic Acids Res. 17: 2919-2932, 1989). Forthe 5′-RACE method, the Marathon cDNA Amplification Kit (CLONTECH) wasused according to the instructions attached to the kit. Using about 1 μgof mRNA prepared as above as a template, the cDNA synthesis primer wasadded, which was reacted with a reverse transcriptase at 42° C. for 60minutes to effect reverse transcription to cDNA.

[0136] This was reacted with DNA polymerase I, DNA ligase, and RNaseH at16° C. for 1.5 hour, and with T4 DNA polymerase at 16° C. for 45 minutesthereby to synthesize a double stranded cDNA. The double stranded cDNAwas extracted with phenol and chloroform, and recovered by ethanolprecipitation. By overnight reaction with T4 DNA ligase at 16° C., acDNA adapter was ligated to both ends of the double stranded cDNA. Thereaction mixture was diluted 50 times with a 10 mM Tricine-KOH (pH 8.5)containing 0.1 mM EDTA. Using this as a template, the gene encoding theL chain V region was amplified by PCR. The adapter primer 1 was used forthe 5′-end primer, and for the 3′-end primer the MKC primer (SEQ ID NO:2) (S. T. Jones, et al., Biotechnology, 9: 88-89, 1991) was used.

[0137] PCR solutions contained, in 100 μl, 120 mM Tris-HCl (pH 8.0), 10mM KCl, 6 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs(dATP, dGTP, dCTP, dTTP), 1 mM MgCl₂, 2.5 units of KOD DNA polymerase(Toyo Boseki), 30-50 pmole of adapter primer 1, as well as MKC primer,and 1 μl of a reaction mixture of cDNA to which the cDNA adapter wasligated.

[0138] All PCRs were carried out using the DNA Thermal Cycler 480(Perkin-Elmer), and the PCR was performed for thirty cycles at atemperature cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and74° C. for 1 minute.

[0139] (3) Purification and Fragmentation of PCR Products

[0140] The above PCR reaction mixture was extracted with phenol andchloroform, and the amplified DNA fragments were recovered by ethanolprecipitation. DNA fragments were digested with the restriction enzymeXmaI (New England Biolabs) at 37° C. for 1 hour. The XmaI-digestionmixture was separated by agarose gel electrophoresis using 2%-3% NuSieveGTG agarose (FMC BioProducts), and the agarose strips containing DNAfragments about 500 bp long as the H chain V region and DNA fragmentsabout 500 bp long as the L chain V region were excised. The agarosestrips were extracted with phenol and chloroform, DNA fragments wereprecipitated with ethanol, which were then dissolved in 10 μl of 10 mMTris-HCl (pH 8.0) containing 1 mM EDTA (hereinafter referred to as TE).

[0141] The XmaI-digested DNA fragments prepared as above containing agenes encoding a mouse H chain V region and L chain V region and thepUC19 plasmid vector prepared by digesting with XmaI were ligated usingthe DNA ligation kit ver.2 (Takara Shuzo) by reacting at 16° C. for 1hour according to the instructions attached to the kit.

[0142] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C.

[0143] Then, 300 μl of the Hi-Competence Broth (Nippongene) was addedthereto, incubated at 37° C. for 1 hour. Then, Escherichia coli wasplated on a LB agar medium (Molecular Cloning: A Laboratory Manual,Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) containing100 μg/ml ampicillin (hereinafter referred to as LBA agar medium), andincubated overnight at 37° C. to obtain an E. coli transformant.

[0144] The transformant was cultured overnight in 3 ml or 4 ml of a LBmedium containing 50 μg/ml ampicillin (hereinafter referred to as LBAmedium) at 37° C., and from the cell fractions, plasmid DNA was preparedusing the QIAprep Spin Plasmid Kit (QIAGEN), and then the nucleotidesequence was determined.

[0145] (4) Determination of the Nucleotide Sequence of the Gene Encodinga Mouse Antibody V Region

[0146] The nucleotide sequence of the cDNA coding region in the aboveplasmid was determined using the Dye Terminator Cycle Sequencing FSReady Reaction Kit (Perkin-Elmer) by the DNA Sequencer 373A(Perkin-Elmer). As the sequencing primer, M13 Primer M4 (Takara Shuzo)(SEQ ID NO: 3) and M13 Primer RV (Takara Shuzo) (SEQ ID NO: 4) wereused, and the sequence was determined by confirming the nucleotidesequence in both directions.

[0147] Thus obtained plasmid containing the gene encoding the mouse Hchain V region derived from the hybridoma ATR-5 was designated asATR-5Hv/pUC19, and the thus obtained plasmid containing the geneencoding a mouse L chain V region derived from the hybridoma ATR-5 wasdesignated as ATR-5Lv/pUC19. The nucleotide sequences of the genesencoding the H chain V region of each mouse antibody contained in theplasmid ATR-5Hv/pUC19 (including the corresponding amino acid sequences)is shown in SEQ ID NO: 5 and 99, respectively, and the nucleotidesequences of the genes encoding the L chain V region of each mouseantibody contained in the plasmid ATR-5Lv/pUC19 (including thecorresponding amino acid sequences) is shown in SEQ ID NO: 6 and 100,respectively.

Reference Example 4 Construction of Chimeric Antibody

[0148] A chimeric ATR-5 antibody was generated in which the mouse ATR-5antibody V region was ligated to the human antibody C region. A chimericantibody expression vector was constructed by ligating the gene encodingthe ATR-5 antibody V region to an expression vector encoding the humanantibody C region.

[0149] (1) Construction of a Chimeric Antibody H Chain V Region

[0150] The ATR-5 antibody H chain V region was modified by the PCRmethod in order to ligate it to an expression vector encoding the humanantibody H chain C region. The 5′-end primer ch5HS (SEQ ID NO: 7) wasdesigned so as to hybridize the 5′-end of DNA encoding the V region andto have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol.196: 947-950, 1987) and a recognition sequence of the restriction enzymeSalI. The 3′-end primer ch5HA (SEQ ID NO: 8) was designed so as tohybridize the 3′-end of DNA encoding the J region and to have arecognition sequence of the restriction enzyme NheI.

[0151] The PCR solutions contained, in 10011,120 mM Tris-HCl (pH 8.0),10 mM KCl, 6 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs(dATP, dGTP, dCTP, dTTP), 1 mM MgCl₂, 2.5 units of KOD DNA polymerase(Toyo Boseki), 50 pmole of the ch5HS primer and the ch5HA primer, aswell as 1 μl of the plasmid ATR5Hv/pUC19 as a template DNA. For PCR, theDNA Thermal Cycler 480 (Perkin-Elmer) was used, and the PCR wasperformed for thirty cycles at a temperature cycle of 94° C. for 30seconds, 55° C. for 30 seconds, and 74° C. for 1 minute.

[0152] The PCR reaction mixture was extracted with phenol andchloroform, and the amplified DNA fragments were recovered by ethanolprecipitation. The DNA fragments were digested with the restrictionenzyme NheI (Takara Shuzo) at 37° C. for 1 hour, and then with therestriction enzyme SalI (Takara Shuzo) at 37° C. for 1 hour. Thedigestion mixture was separated by agarose gel electrophoresis using a3% NuSieve GTG agarose (FMC BioProducts), and the agarose stripscontaining about 450 bp long DNA fragments were excised. The agarosestrips were extracted with phenol and chloroform, and the DNA fragmentswere precipitated with ethanol, which were then dissolved in 20 μl ofTE.

[0153] As the cloning vector, an altered promoter vector (hereinafterreferred to as CVIDEC) was used in which the recognition sequences ofthe restriction enzymes NheI, SalI, and SplI, BglII were introduced. Thegene fragment prepared as above encoding the mouse H chain v region andthe CVIDEC vector prepared by digesting with NheI and SalI were ligatedusing the DNA ligation kit ver.2 (Takara Shuzo) by reacting at 16° C.for 1 hour according to the instructions attached to the kit.

[0154] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of Hi-Competence Broth (Nippongene)was added thereto, incubated at 37° C. for 1 hour, and then the E. coliwas plated on a 100 μg/ml LBA agar medium and incubated overnight at 37°C. to obtain an E. coli transformant. The transformant was culturedovernight at 37° C. in 3 ml of the LBA medium, and from the cellfractions, plasmid DNA was prepared using the QIAprep Spin Plasmid Kit(QIAGEN).

[0155] The nucleotide sequence of the cDNA coding region in the plasmidwas determined using the Dye Terminator Cycle Sequencing FS ReadyReaction Kit (Perkin-Elmer) by the DNA Sequencer 373A (Perkin-Elmer). Asthe sequencing primer, M13 Primer M4 (Takara Shuzo) and M13 Primer RV(Takara Shuzo) were used, and the sequence was determined by confirmingthe nucleotide sequence in both directions. The plasmid that containsthe gene encoding the ATR-5 antibody H chain v region, a SalIrecognition sequence and the Kozak consensus sequence at the 5′-end, anda NheI recognition sequence at the 3′-end was designated aschATR5Hv/CVIDEC.

[0156] (2) Construction of a Chimeric Antibody L Chain V Region

[0157] The ATR-5 antibody L chain V region was modified by the PCRmethod in order to ligate it to an expression vector encoding the humanantibody L chain C region. The 5′-end primer ch5LS (SEQ ID NO: 9) wasdesigned so as to hybridize to the 5′-end of the DNA encoding the Vregion and to have the Kozak consensus sequence (Kozak, M. et al., J.Mol. Biol. 196: 947-950, 1987) and a recognition sequence of therestriction enzyme BglII. The 3′-end primer ch5LA (SEQ ID NO: 10) wasdesigned so as to hybridize to the 3′-end of the DNA encoding the Jregion and to have a recognition sequence of the restriction enzymeSplI.

[0158] The PCR solutions contained, in 10011,120 mM Tris-HCl (pH 8.0),10 mM KCl, 6 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs(dATP, dGTP, dCTP, dTTP), 1 mM MgCl₂, 2.5 units of KOD DNA polymerase(Toyo Boseki), 50 pmole of the ch5LS primer and the ch5LA primer, aswell as 1 μl of the plasmid ATR5Lv/pUC19 as a template DNA. For PCR theDNA Thermal Cycler 480 (Perkin-Elmer) was used, and the PCR wasperformed for thirty cycles at a temperature cycle of 94° C. for 30seconds, 55° C. for 30 seconds, and 74° C. for 1 minute.

[0159] The PCR reaction mixture was extracted with phenol andchloroform, and the amplified DNA fragments were recovered by ethanolprecipitation. The DNA fragments were digested with the restrictionenzyme SplI (Takara Shuzo) at 37° C. for 1 hour, and then with therestriction enzyme BglII (Takara Shuzo) at 37° C. for 1 hour. Thedigestion mixture was separated by agarose gel electrophoresis using a3% NuSieve GTG agarose (FMC BioProducts), and the agarose stripscontaining about 400 bp long DNA fragments were excised. The agarosestrips were extracted with phenol and chloroform, the DNA fragments wereprecipitated with ethanol, which were then dissolved in 20 μl of TE.

[0160] The gene fragment prepared as above encoding the mouse L chain Vregion, and the CVIDEC vector prepared by digesting with SplI and BglII,were ligated using the DNA ligation kit ver.2 (Takara Shuzo) by reactingat 16° C. for 1 hour according to the instructions attached to the kit.

[0161] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of Hi-Competence Broth (Nippongene)was added thereto, incubated at 37° C. for 1 hour, and then the E. coliwas plated on a 100 μg/ml LBA agar medium and incubated overnight at 37°C. to obtain an E. coli transformant. The transformant was culturedovernight at 37° C. in 3 ml of the LBA medium and, from the cellfractions, plasmid DNA was prepared using the QIAprep Spin Plasmid Kit(QIAGEN).

[0162] The nucleotide sequence of the cDNA coding region in the plasmidwas determined using the Dye Terminator Cycle Sequencing FS ReadyReaction Kit (Perkin-Elmer) by the DNA Sequencer 373A (Perkin-Elmer). Asthe sequencing primer, M13 Primer M4 (Takara Shuzo) and M13 Primer RV(Takara Shuzo) were used, and the sequence was determined by confirmingthe nucleotide sequence in both directions. The plasmid that containsthe gene encoding the ATR-5 antibody L chain V region and that has aBglII recognition sequence and the Kozak consensus sequence at the5′-end and a SplI recognition sequence at the 3′-end was designated aschATR5Lv/CVIDEC.

[0163] (3) Construction of a Chimeric Antibody Expression Vector

[0164] A chimeric antibody expression vector was constructed using anantibody expression vector introduced from IDEC Pharmaceuticals. As thevector, the IgG1-type antibody expression vector N5KG1(V) and theIgG4-type antibody expression vector N5KG4P were used. The chimericATR-5 antibody expression vector was generated by ligating a geneencoding the H chain V region of ATR-5 to the SalI-NheI site locatedimmediately before the human antibody H chain C region of the expressionvector N5KG1(V) or N5KG4P and ligating a gene encoding the L chain Vregion of ATR-5 to the BglII-SplI site located immediately before thehuman antibody L chain C region of the expression vector N5KG1(V) orN5KG4P.

[0165] (i) Introduction of H Chain V Region

[0166] The plasmid chATR5Hv/CVIDEC was digested with the restrictionenzyme NheI (Takara Shuzo) at 37° C. for 3 hours, and with therestriction enzyme SalI (Takara Shuzo) at 37° C. for 3 hours. Thedigestion mixture was separated by agarose gel electrophoresis using1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose stripscontaining about 450 bp long DNA fragments were excised. The agarosestrips were extracted with phenol and chloroform, and the DNA fragmentswere precipitated with ethanol, which were then dissolved in 20 μl ofTE.

[0167] The expression vector N5KG1(V) and N5KG4P were digested with therestriction enzyme NheI (Takara Shuzo) at 37° C. for 3 hours, and withthe restriction enzyme SalI (Takara Shuzo) at 37° C. for 3 hours. Thedigestion mixture was separated by agarose gel electrophoresis using1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose stripscontaining about 9000 bp long DNA fragments were excised. The agarosestrips were extracted with phenol and chloroform, and the DNA fragmentswere precipitated with ethanol, which were then dissolved in 60 μl ofTE.

[0168] The SalI-NheI DNA fragment prepared as above containing the geneencoding the H chain V region and N5KG1(V) or N5KG4P digested with SalIand NheI were ligated using the DNA ligation kit ver.2 (Takara Shuzo) byreacting at 16° C. for 1 hour according to the attached instructions.

[0169] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of Hi-Competence Broth (Nippongene)was added thereto, incubated at 37° C. for 1 hour, and then the E. coliwas plated on a 100 μg/ml LBA agar medium and incubated overnight at 37°C. to obtain an E. coli transformant. The transformant was culturedovernight at 37° C. in 3 ml of the LBA medium, and from the cellfractions, plasmid DNA was prepared using the QIAprep Spin Plasmid Kit(QIAGEN). These plasmids containing the genes encoding the chimericATR-5 antibody H chain were designated as chATR5Hv/N5KG1(V) andchATR5Hv/N5KG4P, respectively.

[0170] (ii) Introduction of the L Chain V Region

[0171] The plasmid chATR5Lv/CVIDEC was digested with the restrictionenzymes BglII (Takara Shuzo) and SplI (Takara Shuzo) at 37° C. for 1.5hour. The digestion mixture was separated by agarose gel electrophoresisusing 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose stripscontaining about 400 bp long DNA fragments were excised. The agarosestrips were extracted with phenol and chloroform, and the DNA fragmentswere precipitated with ethanol, which were then dissolved in 20 μl ofTE.

[0172] The plasmids chATR5Hv/N5KG1(V) and chATR5Hv/N5KG4P were digestedwith the restriction enzymes BglII (Takara Shuzo) and SplI (TakaraShuzo) at 37° C. for 1.5 hour. The digestion mixture was separated byagarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMCBioProducts), and the agarose strips containing about 9400 bp long DNAfragments were excised. The agarose strips were extracted with phenoland chloroform, DNA fragments were precipitated with ethanol, which werethen dissolved in 20 μl of TE.

[0173] The SplI-BglII DNA fragment prepared as above containing the geneencoding the L chain V region and chATR5Hv/N5KG1(V) or chATR5Hv/N5KG4Pdigested with SplI and BglII were ligated using the DNA ligation kitver.2 (Takara Shuzo) by reacting at 16° C. for 1 hour according to theattached instructions.

[0174] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of Hi-Competence Broth (Nippongene)was added thereto, incubated at 37° C. for 1 hour, and then the E. coliwas plated on a 1001g/ml LBA agar medium and incubated overnight at 37°C. to obtain an E. coli transformant. The transformant was culturedovernight at 37° C. in 1 liter of the 2xYT medium containing 50 μg/mlampicillin, and from the cell fractions, plasmid DNA was prepared usingthe Plasmid Maxi Kit (QIAGEN). These plasmids containing the geneencoding the chimeric ATR-5 antibody were designated as chATR5/N5KG1(V)and chATR5/N5KG4P, respectively.

[0175] (4) Transfection into COS-7 Cells

[0176] In order to evaluate the activity of binding to the antigen andthe neutralizing activity of chimeric antibody, the above expressionplasmid was transfected to COS-7 cells and the antibody was transientlyexpressed.

[0177] The plasmid chATR5/N5KG1(V) or chATR5/N5KG4P was transduced intoCOS-7 cells by electroporation using a Gene Pulser instrument (Bio Rad).Fifty μg of the plasmid was added to 0.78 ml of the COS-7 cellssuspended in the Dulbecco PBS (−) (hereinafter referred to as PBS) at acell concentration of 1×10⁷ cells/ml, which was subjected to pulses of1,500 V and 25 μF capacity.

[0178] After 10 minutes of the recovery period at room temperature, theelectroporated cells were suspended in a DMEM medium containing 5% Ultralow IgG fetal bovine serum (GIBCO), and cultured using a 10 cm culturedish in a 5% CO₂ incubator. After culturing for 24 hours, the culturesupernatant was aspirated off and, then, a serum-free medium HBCHO(Irvine Scientific) was added. After further culturing for 72 hours, theculture supernatant was collected and centrifuged to remove cell debris.

[0179] (5) Purification of Antibody

[0180] From the culture supernatant of the COS-7 cells, chimericantibody was purified using rprotein A Sepharose Fast Flow (PharmaciaBiotech) as follows.

[0181] One ml of rprotein A Sepharose Fast Flow was filled into a columnand the column was equilibrated by 10 volumes of TBS. The culturesupernatant of COS-7 cells was applied to the equilibrated column, whichwas then washed with 10 volumes of TBS.

[0182] The adsorbed antibody fraction was then eluted by 13.5 ml of 2.5mM HCl (pH 3.0), and the eluate was immediately neutralized by adding1.5 ml of 1 M Tris-HCl (pH 8.0).

[0183] By performing ultrafiltration twice for the purified antibodyfraction using the Centriprep 100 (Amicon), the solvent was replacedwith 50 mM Tris-HCl (pH 7.6) containing 150 mM NaCl (hereinafterreferred to as TBS), and was finally concentrated to about 1.5 ml.

[0184] (6) Establishment of a Stably-Producing CHO Cell Line

[0185] In order to establish a cell line that stably produces chimericantibody, the above expression plasmid was introduced into CHO cells(DG44) acclimated to the CHO-S-SFMII serum-free medium (GIBCO).

[0186] The plasmid chATR5/N5KG1(V) or chATR5/N5KG4P was cleaved with therestriction enzyme SspI (Takara Shuzo) to linearize DNA, and afterextraction with phenol and chloroform, DNA was recovered by ethanolprecipitation. The linearized plasmid was transduced into the DG44 cellsby electroporation using a Gene Pulser instrument (Bio Rad). Ten μg ofthe plasmid was added to 0.78 ml of DG44 cells suspended in PBS at acell concentration of 1×10⁷ cells/ml, which was subjected to pulses of1,500 V and 25 μF capacity.

[0187] After a 10 minute recovery period at room temperature, theelectroporated cells were suspended in a CHO-S-SFMII medium (GIBCO)containing hypoxanthine/thymidine (GIBCO), and cultured using two96-well plates (Falcon) in a 5% CO₂ incubator. On the day after thestart of culturing, the medium was changed to a selection mediumcontaining the CHO-S-SFMII medium (GIBCO) containinghypoxanthine/thymidine (GIBCO) and 500 μg/ml GENETICIN (G418Sulfate,GIBCO) to select cells into which the antibody gene had been introduced.About two weeks after changing the selection medium, the cells wereexamined under a microscope. After favorable cell growth was observed,the amount of antibody produced was measured by the ELISA describedbelow for determining antibody concentration, and cells having a highproduction yield of antibody were selected.

Reference Example 5 Construction of Humanized Antibody

[0188] (1) Construction of Humanized Antibody H Chain

[0189] (i) Construction of the Humanized H Chain Version “a”

[0190] Humanized ATR-5 antibody H chain was generated using CDR-graftingby the PCR method. In order to generate the humanized antibody H chainversion “a” having the FRs derived from human antibody L39130 (DDBJ, GaoL. et al., unpublished, 1995), seven PCR primers were used. TheCDR-grafting primers hR5Hv1S (SEQ ID NO: 11), hR5Hv2S (SEQ ID NO: 12),and hR5Hv4S (SEQ ID NO: 13) have a sense DNA sequence, and the CDRgrafting primers hR5Hv3A (SEQ ID NO: 14) and hR5Hv5A (SEQ ID NO: 15)have an antisense DNA sequence, each primer having a 18-35 bpcomplementary sequence on both ends thereof.

[0191] hR5Hv1S was designed to have the Kozak consensus sequence (Kozak,M. et al., J. Mol. Biol. 196: 947-950, 1987) and a SalI recognitionsite, and hR5Hv5A was designed to have a NheI recognition site. Theexogenous primer hR5HvPrS (SEQ ID NO: 16) has a homology with theCDR-grafting primer hR5Hv1S, and hR5HvPrA (SEQ ID NO: 17) has a homologywith the CDR-grafting primer hR5Hv5A.

[0192] The CDR-grafting primers hR5Hv1S, hR5Hv2S, hR5Hv3A, hR5Hv4S, andhR5Hv5A, and exogenous primers hR5HvPrS and hR5HvPrA were synthesizedand purified by Pharmacia Biotech.

[0193] PCR was performed using the KOD DNA polymerase (Toyo Boseki) andusing the attached buffer under the condition of containing 120 mMTris-HCl (pH 8.0), 10 mM KCl, 6 mM (NH₄)₂SO₄, 0.1% Triton X100, 0.001%BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 mM MgCl₂, 2.5 units of KODDNA polymerase (Toyo Boseki), and 5 pmole each of the CDR-graftingprimers hR5Hv1S, hR5Hv2S, hR5Hv3A, hR5Hv4S, and hR5Hv5A in 98 μl, for 5cycles at a temperature cycle of 94° C. for 30 seconds, 50° C. for 1minute, and 72° C. for 1 minute. After further addition of 100 pmole ofexogenous primers hR5HvPrS and hR5HvPrA, PCR was performed for 25 cyclesin a system of 100 μl with the same temperature cycle. DNA fragmentsamplified by the PCR method were separated by agarose gelelectrophoresis using a 2% NuSieve GTG agarose (FMC BioProducts).

[0194] The agarose strips containing about 430 bp long DNA fragmentswere excised, to which 3 volumes (ml/g) of TE was added, and then wereextracted with phenol, phenol/chloroform, and chloroform to purify theDNA fragments. After precipitating the purified DNA with ethanol, onethird the volume thereof was dissolved in 17 μl of water. The PCRreaction mixture obtained was digested with NheI and SalI, and wasligated to the plasmid vector CVIDEC prepared by digesting with NheI andSalI, using the DNA ligation kit ver.2 (Takara Shuzo) according to theinstructions attached to the kit.

[0195] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of the Hi-Competence Broth(Nippongene) was added thereto, incubated at 37° C. for 1 hour, and thenthe E. coli was plated on a 100 μg/ml LBA agar medium and incubatedovernight at 37° C. to obtain an E. coli transformant. The transformantwas cultured overnight at 37° C. in 3 ml of the LBA medium, and from thecell fractions, plasmid DNA was prepared using the QIAprep Spin PlasmidKit (QIAGEN).

[0196] The nucleotide sequence of the cDNA coding region in the plasmidwas determined using the Dye Terminator Cycle Sequencing FS ReadyReaction Kit (Perkin-Elmer) by the DNA Sequencer 373A (Perkin-Elmer). Asthe sequencing primer, M13 Primer M4 (Takara Shuzo) and M13 Primer RV(Takara Shuzo) were used, and the sequence was determined by confirmingthe nucleotide sequence in both directions.

[0197] Since mutation and/or deletion were observed before or after theEcoT221 recognition site, each of fragments having the correct sequencewas ligated and then subcloned again to CVIDEC to determine thenucleotide sequence. The plasmid having the correct sequence wasdesignated as hATR5Hva/CVIDEC. The nucleotide sequence and thecorresponding amino acid sequence of the humanized H chain version “a”contained in the plasmid hATR5Hva/CVIDEC are shown in SEQ ID NO: 18. Theamino acid sequence of version “a” is also shown in SEQ ID NO: 19.

[0198] (ii) Construction of Humanized H Chain Versions “b” and “c”

[0199] Versions “b” and “c” were generated by replacing the FR3 ofversion “a” with the FR3 derived from another human antibody using theFR-shuffling method. In order to replace the FR3 in version “b” with onederived from human antibody Z34963 (DDBJ, Borretzen M. et al., Proc.Natl. Acad. Sci. USA, 91: 12917-12921, 1994), the four DNA primersencoding the FR3 were generated. The FR-shuffling primers F3RFFS (SEQ IDNO: 20) and F3RFBS (SEQ ID NO: 21) have a sense DNA sequence and F3RFFA(SEQ ID NO: 22) and F3RFBA (SEQ ID NO: 23) have an antisense DNAsequence. F3RFFS and F3RFFA have a sequence complementary to each other,and have BalI and XhoI recognition sequences on both ends. F3RFBS andF3RFBA have a sequence complementary to each other, and have XhoI andNcoI recognition sequences on both ends.

[0200] In order to replace the FR3 in version “c” with one derived fromhuman antibody P01825 (SWISS-PROT, Poljak RJ. et al., Biochemistry, 16:3412-3420, 1977), four DNA primers encoding the FR3 were generated. TheFR-shuffling primers F3NMFS (SEQ ID NO: 24) and F3NMBS (SEQ ID NO: 25)have a sense DNA sequence and F3NMFA (SEQ ID NO: 26) and F3NMBA (SEQ IDNO: 27) have an antisense DNA sequence. F3RFBS and F3RFBA have asequence complementary to each other, and have BalI and XhoI recognitionsequences on both ends.

[0201] F3RFFS, F3RFBS, F3RFFA, F3RFBA, F3NMFS, F3NMBS, F3NMFA, andF3NMBA were synthesized by Pharmacia Biotech. F3RFFS and F3RFFA, andF3RFBS and F3RFBA were annealed, and were digested with BalI and XhoI,and NcoI and XhoI, respectively. They were introduced to the plasmidhATR5Hva/CVIDEC (BalI/NcoI) prepared by digesting with BalI and NcoI,and the nucleotide sequence was determined. The plasmid having thecorrect sequence was designated as hATR5Hvb/CVIDEC. The nucleotidesequence and the corresponding amino acid sequence of the humanized Hchain version “b” contained in the plasmid hATR5Hvb/CVIDEC are shown inSEQ ID NO: 28. The amino acid sequence of version “b” is also shown inSEQ ID NO: 29.

[0202] F3NMFS and F3NMFA, and F3NMBS and F3NMBA were annealed, and weredigested with BalI and XhoI, and NcoI and XhoI, respectively. They wereintroduced to the plasmid hATR5Hva/CVIDEC (BalI/NcoI) prepared bydigesting with BalI and NcoI, and the nucleotide sequence wasdetermined. The plasmid having the correct sequence was designated ashATR5Hvc/CVIDEC. The nucleotide sequence and the corresponding aminoacid sequence of the humanized H chain version “c” contained in theplasmid hATR5Hvc/CVIDEC are shown in SEQ ID NO: 30. The amino acidsequence of version “c” is also shown in SEQ ID NO: 31.

[0203] (iii) Construction of Humanized H Chain Versions “d” and “e”

[0204] Versions “d” and “e” were generated by replacing the FR3 ofversion “a” with the FR3 derived from another human antibody using theFR-shuffling method. In order to replace the FR3 in version “d” with onederived from human antibody M62723 (DDBJ, Pascual V. et al., J. Clin.Invest., 86: 1320-1328, 1990), four DNA primers encoding the FR3 weregenerated. The FR-shuffling primer F3EPS (SEQ ID NO: 32) has a sense DNAsequence and F3EPA (SEQ ID NO: 33) has an antisense DNA sequence, andthe 3′-end of the primers has a complementary sequence of 18 bp.

[0205] Exogenous primers F3PrS (SEQ ID NO: 34) and F3PrA (SEQ ID NO: 35)have a homology with the FR-shuffling primers F3EPS and F3EPA, and canalso be used for other FR3 FR-shuffling. In order to replace the FR3 inversion “e” with one derived from the human antibody Z80844 (DDBJ,Thomsett AR. et al., unpublished), two DNA primers encoding the FR3 weregenerated. The FR-shuffling primers F3VHS (SEQ ID NO: 36) has a senseDNA sequence and F3VHA (SEQ ID NO: 37) has an antisense DNA sequence,and the 3′-end of the primers has a complementary sequence of 18 bp.F3EPS, F3EPA, F3PrS, F3PrA, F3VHS and F3VHA were synthesized byPharmacia Biotech.

[0206] PCR was performed using the KOD DNA polymerase (Toyo Boseki)using the attached buffer under the condition of containing 5 μl each of1 μM FR-shuffling primers F3EPS and F3EPA, or F3VHS and F3VHA, 0.2 mMdNTPs, 1.0 mM MgCl₂, and 2.5 units of KOD DNA polymerase in 100 μl ofthe reaction mixture, for 5 cycles at a temperature cycle of 94° C. for30 seconds, 50° C. for 1 minute, and 74° C. for 1 minute. After furtheraddition of 100 pmole of exogenous primers F3PrS and F3PrA, PCR wasperformed for 25 cycles with the same temperature cycle.

[0207] DNA fragments amplified by the PCR method were separated byagarose gel electrophoresis using a 2% Nu Sieve GTG agarose (FMCBioProducts). The agarose strips containing about 424 bp long DNAfragments were excised, to which 3 volumes (ml/g) of TE was added, andthen were extracted with phenol, phenol/chloroform, and chloroform topurify the DNA fragments. After precipitating the purified DNA withethanol, one third the volume thereof was dissolved in 14 μl of water.The PCR reaction mixture obtained was digested with BalI and NcoI, andwas introduced to the plasmid hATR5Hva/CVIDEC (BalI/NcoI) prepared bydigesting with BalI and NcoI, and the nucleotide sequence wasdetermined.

[0208] The plasmids having the correct sequence were designated ashATR5Hvd/CVIDEC and hATR5Hve/CVIDEC. The nucleotide sequence and thecorresponding amino acid sequence of the humanized H chain version “d”contained in the plasmid hATR5Hvd/CVIDEC are shown in SEQ ID NO: 38, andthe amino acid sequence of version “d” is also shown in SEQ ID NO: 39.The nucleotide sequence and the corresponding amino acid sequence of thehumanized H chain version “e” contained in the plasmid hATR5Hve/CVIDECare shown in SEQ ID NO: 40, and the amino acid sequence of version “e”is also shown in SEQ ID NO: 41.

[0209] (iv) Construction of Humanized H Chain Versions “f” and “g”

[0210] Versions “f” and “g” were generated by replacing the FR3 ofversion “a” with the FR3 derived from another human antibody using theFR-shuffling method. In order to replace the FR3 in version “f” with onederived from human antibody L04345 (DDBJ, Hillson J L. et al., J. Exp.Med., 178: 331-336, 1993) and to replace the FR3 in version “g” with onederived from human antibody S78322 (DDBJ, Bejcek BE. et al., CancerRes., 55: 2346-2351, 1995), two primers each encoding the FR3 weresynthesized. The FR-shuffling primer F3SSS (SEQ ID NO: 42) of version“f” has a sense DNA sequence and F3SSA (SEQ ID NO: 43) has an antisenseDNA sequence, and the 3′-end of the primers has a complementary sequenceof 18 bp.

[0211] F3CDS (SEQ ID NO: 44) of version “g” has a sense DNA sequence andF3CDA (SEQ ID NO: 45) has an antisense DNA sequence, and the 3′-end ofthe primers has a complementary sequence of 18 bp. F3SSS, F3SSA, F3CDS,and F3CDA were synthesized and purified by Pharmacia Biotech. PCR wasperformed using the KOD DNA polymerase (Toyo Boseki) using the attachedbuffer under the condition of containing 5 μl each of 1 μM FR-shufflingprimers F3SSS and F3SSA, or F3CDS and F3CDA, 0.2 mM dNTPs, 1.0 mM MgCl₂,and 2.5 units of KOD DNA polymerase in 100 μl of the reaction mixture,for 5 cycles at a temperature cycle of 94° C. for 30 seconds, 50° C. for1 minute, and 74° C. for 1 minute. After further addition of 100 pmoleof exogenous primers F3PrS and F3PrA, PCR was performed for 25 cycleswith the same temperature cycle.

[0212] DNA fragments amplified by the PCR method were separated byagarose gel electrophoresis using a 2% NuSieve GTG agarose (FMCBioProducts). The agarose strips containing about 424 bp long DNAfragments were excised, to which 3 volumes (ml/g) of TE was added, andthen were extracted with phenol, phenol/chloroform, and chloroform topurify the DNA fragments. After precipitating the purified DNA withethanol, one third the volume thereof was dissolved in 14 μl of water.The PCR reaction mixture obtained was digested with BalI and NcoI, andwas introduced to the plasmid hATR5Hva/CVIDEC (BalI/NcoI) prepared bydigesting with BalI and NcoI, and the nucleotide sequence wasdetermined.

[0213] The plasmids having the correct sequence were designated ashATR5Hvf/CVIDEC and hATR5Hvg/CVIDEC. The nucleotide sequence and thecorresponding amino acid sequence of the humanized H chain version “f”contained in the plasmid hATR5Hvf/CVIDEC, and the amino acid sequence ofversion “f” are shown in SEQ ID NO: 46 and 47. The nucleotide sequenceand the corresponding amino acid sequence of the humanized H chainversion “g” contained in the plasmid hATR5Hvg/CVIDEC, and the amino acidsequence of version “g” are shown in SEQ ID NO: 48 and 49.

[0214] (v) Construction of the Humanized H Chain Version

[0215] Version “h” was generated by replacing the FR3 of version “a”with the FR3 derived from another human antibody using the FR-shufflingmethod. In order to replace the FR3 in version “h” with one derived fromthe human antibody z26827 (DDBJ, van Der Stoep et al., J. Exp. Med.,177: 99-107, 1993), two primers each encoding the FR3 were synthesized.The FR-shuffling primer F3ADS (SEQ ID NO: 50) of version “h” has a senseDNA sequence and F3ADA (SEQ ID NO: 51) has an antisense DNA sequence,and the 3′-end of the primers has a complementary sequence of 18 bp.

[0216] F3ADS and F3ADA were synthesized and purified by PharmaciaBiotech. PCR was performed using the KOD DNA polymerase (Toyo Boseki)using the attached buffer under the condition of containing 5 μl each of1 μM FR-shuffling primers F3ADS and F3ADA, 0.2 mM dNTPs, 1.0 mM MgCl₂,and 2.5 units of KOD DNA polymerase in 100 μl of the reaction mixture,for 5 cycles at a temperature cycle of 94° C. for 30 seconds, 50° C. for1 minute, and 74C for 1 minute. After further addition of 100 pmole ofexogenous primers F3PrS and F3PrA, PCR was performed for 25 cycles withthe same temperature cycle. DNA fragments amplified by the PCR methodwere separated by agarose gel electrophoresis using a 2% NuSieve GTGagarose (FMC BioProducts).

[0217] The agarose strips containing about 424 bp long DNA fragmentswere excised, to which 3 volumes (ml/g) of TE was added, and then wereextracted with phenol, phenol/dhloroform, and chloroform to purify theDNA fragments. After precipitating the purified DNA with ethanol, onethird the volume thereof was dissolved in 14 μl of water. The PCRreaction mixture obtained was digested with BalI and NcoI, and wasintroduced to the plasmid hATR5Hva/CVIDEC (BalI/NcoI) prepared bydigesting with BalI and NcoI, and the nucleotide sequence wasdetermined. The plasmids having the correct sequence were designated ashATR5Hvh/CVIDEC. The nucleotide sequence and the corresponding aminoacid sequence of the humanized H chain version “h” contained in theplasmid hATR5Hvh/CVIDEC, and the amino acid sequence of version “h” areshown in SEQ ID NO: 52. The amino acid sequence of version “h” is shownin SEQ ID NO: 53.

[0218] (vi) Construction of Humanized H Chain Versions “i” and “j”

[0219] Versions “i” and “j” were generated by replacing the FR3 ofversion “a” with the FR3 derived from another human antibody using theFR-shuffling method. In order to replace the FR3 in version “i” with onederived from the human antibody U95239 (DDBJ, Manheimer-Lory AAJ.,unpublished) and to replace the FR3 in version “j” with one derived fromthe human antibody L03147 (DDBJ, Collect TA. et al., Proc. Natl. Acad.Sci. USA, 89: 10026-10030, 1992), two primers each encoding the FR3 weresynthesized. The FR-shuffling primer F3MMS (SEQ ID NO: 54) of version“i” has a sense DNA sequence and F3MMA (SEQ ID NO: 55) has an antisenseDNA sequence, and the 3′-end of the primers has a complementary sequenceof 18 bp.

[0220] F3BMS (SEQ ID NO: 56) of version “j” has a sense DNA sequence andF3BMA (SEQ ID NO: 57) has an antisense DNA sequence, and the 3′-end ofthe primers has a complementary sequence of 18 bp. F3MMS, F3MMA, F3BMS,and F3BMA were synthesized and purified by Pharmacia Biotech. PCR wasperformed using the Ampli Taq Gold (Perkin-Elmer) using the attachedbuffer under the condition of containing 5 μl each of 1 μM FR-shufflingprimers F3MMS and F3MMA, or F3BMS and F3BMA, 0.2 mM dNTPs, 1.0 mM MgCl₂,and 2.5 units of Ampli Taq Gold in 100 μl of the reaction mixture, for 5cycles at a temperature cycle of 94° C. for 30 seconds, 50° C. for 1minute, and 74° C. for 1 minute. After further addition of 100 pmole ofexogenous primers F3PrS and F3PrA, PCR was performed for 25 cycles withthe same temperature cycle.

[0221] DNA fragments amplified by the PCR method were separated byagarose gel electrophoresis using a 2% Nu Sieve GTG agarose (FMCBioProducts). The agarose strips containing about 424 bp long DNAfragments were excised, to which 3 volumes (ml/g) of TE was added, andthen were extracted with phenol, phenol/chloroform, and chloroform topurify the DNA fragments. After precipitating the purified DNA withethanol, one third the volume thereof was dissolved in 14 μl of water.The PCR reaction mixture obtained was digested with BalI and NcoI, andwas introduced to the plasmid hATR5Hva/CVIDEC (BalI/NcoI) prepared bydigesting with BalI and NcoI, and the nucleotide sequence wasdetermined.

[0222] The plasmids having the correct sequence were designated ashATR5Hvi/CVIDEC and hATR5Hvj/CVIDEC. The nucleotide sequence and thecorresponding amino acid sequence of the humanized H chain version “i”contained in the plasmid hATR5Hvi/CVIDEC, and the amino acid sequence ofversion “i” are shown in SEQ ID NO: 58 and 59. The nucleotide sequenceand the corresponding amino acid sequence of the humanized H chainversion “j” contained in the plasmid hATR5Hvj/CVIDEC, and the amino acidsequence of version “j” are shown in SEQ ID NO: 60 and 61.

[0223] (vii) Construction of Humanized H Chain Versions “b1” and “d1”

[0224] Versions “b1” and “d1” were generated by replacing the FR2 ofversions “b” and “d” with the FR2 derived from another human antibodyusing the FR-shuffling method. In order to replace the FR2 with onederived from the human antibody P01742 (SWISS-PROT, Cunningham BA. etal., Biochemistry, 9: 3161-3170, 1970), two DNA primers encoding the FR2were synthesized. The FR-shuffling vector F2 MPS (SEQ ID NO: 62) has asense DNA sequence and F2 MPA (SEQ ID NO: 63) has an antisense DNAsequence. They also have a sequence complementary to each other, andhave recognition sequences of EcoT221 and BalI on both ends thereof.

[0225] F2 MPS and F2 MPA were synthesized and purified by PharmaciaBiotech. F2 MPS and F2 MPA were annealed and were digested with EcoT22Iand BalI. They were introduced to plasmids hATR5Hvb/CVIDEC(EcoT22I/BalI) and hATR5Hvd/CVIDEC (EcoT22I/BalI) prepared by digestingwith EcoT22I and BalI, and the nucleotide sequence was determined. Theplasmids having the correct sequence were designated as hATR5Hvb1/CVIDECand hATR5Hvd1/CVIDEC. The nucleotide sequence and the correspondingamino acid sequence of the humanized H chain version “b1” contained inthe plasmid hATR5Hvb1/CVIDEC, and the amino acid sequence of version “b”are shown in SEQ ID NO: 64 and 65. The nucleotide sequence and thecorresponding amino acid sequence of the humanized H chain version “d1”contained in the plasmid hATR5Hvdl/CVIDEC, and the amino acid sequenceof version “d1” are shown in SEQ ID NO: 66 and 67.

[0226] (viii) Construction of Humanized H Chain Versions “b3” and “d3”

[0227] Versions “b3” and “d3” were generated by replacing the FR2 ofversions “b” and “d” with the FR2 derived from another human antibodyusing the FR-shuffling method. In order to replace the FR2 with onederived from the human antibody Z80844 (DDDJ, Thomsett AR. et al.,unpublished), two DNA primers encoding the FR2 were synthesized. TheFR-shuffling vector F2VHS (SEQ ID NO: 68) has a sense DNA sequence andF2VHA (SEQ ID NO: 69) has an antisense DNA sequence. They also have asequence complementary to each other, and have recognition sequences ofEcoT221 and BalI on both ends thereof. The synthesis and purification ofF2VHS and F2VHA was referred to Pharmacia Biotech.

[0228] F2VHS and F2VHA were annealed and were digested with EcoT22I andBalI. They were introduced to plasmids hATR5Hvb/CVIDEC (EcoT22I/BalI)and hATR5Hvd/CVIDEC (EcoT22I/BalI) prepared by digesting with EcoT22Iand BalI, and the nucleotide sequence was determined. The plasmidshaving the correct sequence were designated as hATR5Hvb3/CVIDEC andhATR5Hvd3/CVIDEC. The nucleotide sequence and the corresponding aminoacid sequence of the humanized H chain version “b3” contained in theplasmid hATR5Hvb3/CVIDEC, and the amino acid sequence of version “b3”are shown in SEQ ID NO: 70 and 71. The nucleotide sequence and thecorresponding amino acid sequence of the humanized H chain version “d3”contained in the plasmid hATR5Hvd3/CVIDEC, and the amino acid sequenceof version “d3” are shown in SEQ ID NO: 72 and 73.

[0229] (2) Construction of a Humanized Antibody L Chain V Region

[0230] (i) Version “a”

[0231] The humanized ATR-5 antibody L chain V region was generated bythe CDR-grafting using the PCR method. For the generation of a humanizedantibody L chain (version “a”) having framework regions derived fromhuman antibody Z37332 (DDBJ, Welschof M. et al., J. Immunol. Methods,179: 203-214, 1995), seven PCR primers were used.

[0232] CDR-grafting primers h5Lv1S (SEQ ID NO: 74) and h5Lv4S (SEQ IDNO: 75) have a sense DNA sequence, CDR-grafting primers h5Lv2A (SEQ IDNO: 76), h5Lv3A (SEQ ID NO: 77), and h5Lv5A (SEQ ID NO: 78) have anantisense DNA sequence, and each primer has 20 bp complementarysequences on both ends thereof. Exogenous primers h5LvS (SEQ ID NO: 79)and h5LvA (SEQ ID NO: 80) have a homology with CDR-grafting primersh5Lv1S and h5Lv5A. The synthesis and purification of CDR-graftingprimers h5Lv1S, h5Lv4S, h5Lv2A, h5Lv3A, h5Lv5A, h5LvS, and h5LvA werereferred to Pharmacia Biotech.

[0233] The PCR solutions contain, in 100 μl, 120 mM Tris-HCl (pH 8.0),10 mM KCl, 6 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs(dATP, dGTP, dCTP, dTTP), 1 mM MgCl₂, 2.5 units of KOD DNA polymerase(Toyo Boseki), 50 pmole of the CDR-grafting primers h5Lv1S, h5Lv2A,h5Lv3A, h5Lv4S, and h5Lv5A.

[0234] PCR was performed using the DNA Thermal Cycler 480 (Perkin-Elmer)for 5 cycles with the temperature cycle of 94° C. for 30 seconds, 50° C.for 1 minute, and 72° C. for 1 minute to assemble 5 CDR-graftingprimers. After further addition of 100 pmole of exogenous primers h5LvSand h5LvA to the reaction mixture, PCR was performed for 30 cycles withthe temperature cycle of 94° C. for 30 seconds, 52° C. for 1 minute, and72° C. for 1 minute to amplify the assembled DNA fragments.

[0235] The PCR reaction mixture was separated by agarose gelelectrophoresis using a 3% NuSieve GTG agarose (FMC BioProducts), andthe agarose strips containing about 400 bp long DNA fragments wereexcised. The agarose strips were extracted with phenol and chloroform,DNA fragments were recovered by ethanol precipitation. The recovered DNAfragments were digested with the restriction enzymes SplI (Takara Shuzo)and BglII (Takara Shuzo) at 37° C. for 4 hours. The digestion mixturewas extracted with phenol and chloroform, and after the ethanolprecipitation of the DNA fragments, they were dissolved in 10 μl of TE.The SplI-BglII DNA fragment prepared as above encoding the humanized Lchain V region and the CVIDEC vector prepared by digesting with SplI andBglII were ligated using the DNA ligation kit ver.2 (Takara Shuzo) byreacting at 16° C. for 1 hour according to the instructions attached tothe kit.

[0236] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of Hi-Competence Broth (Nippongene)was added thereto, incubated at 37° C. for 1 hour, and then the E. coliwas plated on a 100 μg/ml LBA agar medium and incubated overnight at 37°C. to obtain an E. coli transformant. The transformant was culturedovernight in 3 ml of the LBA medium, and from the cell fractions,plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN).

[0237] The nucleotide sequence of the cDNA coding region in the plasmidwas determined using the Dye Terminator Cycle Sequencing FS ReadyReaction Kit (Perkin-Elmer) by the DNA Sequencer 373A (Perkin-Elmer). Asthe sequencing primer, M13 Primer M4 (Takara Shuzo) and M13 Primer RV(Takara Shuzo) were used, and the sequence was determined by confirmingthe nucleotide sequence in both directions. The plasmid that containsthe gene encoding the humanized antibody L chain V region and that has aBglII recognition sequence and the Kozak sequence at the 5′-end, and aSplI recognition sequence at the 3′-end was designated ashATR5Lva/CVIDEC. The nucleotide sequence (including the correspondingamino acid sequence) of the humanized L chain version “a” is shown inSEQ ID NO: 81. The amino acid sequence of version “a” is also shown inSEQ ID NO: 82.

[0238] (ii) Versions “b” and “c”

[0239] Versions “b” and “c” were generated by replacing (FR-shuffling)the FR3 of version “a”. For version “b” the FR3 derived from humanantibody S68699 (DDBJ, Hougs L. et al., Exp. Clin. Immunogen et., 10:141-151, 1993) was used, and for version “c1” the FR3 derived from humanantibody P01607 (SWISS-PROT, Epp O et al., Biochemistry, 14: 4943-4952,1975) was used, respectively.

[0240] Primers F3SS (SEQ ID NO: 83) and F3SA (SEQ ID NO: 84) encodingthe FR3 of version “b”, or primers F3RS (SEQ ID NO: 85) and F3RA (SEQ IDNO: 86) encoding the FR3 of version “c” have a sequence complementary toeach other, and have the recognition sequences of the restrictionenzymes KpnI and PstI on both ends thereof. The synthesis andpurification of F3SS, F3SA, F3RS, and F3RA were referred to PharmaciaBiotech. 100 pmole each of F3SS and F3SA, or F3RS and F3RA were annealedby treating at 96° C. for 2 minutes and at 50° C. for 2 minutes and thedouble stranded DNA fragments were generated.

[0241] These double stranded DNA fragments were digested with therestriction enzyme KpnI (Takara Shuzo) at 37° C. for 1 hour, and thenwith the restriction enzyme PstI (Takara Shuzo) at 37° C. for 1 hour.The digestion mixture was extracted with phenol and chloroform and,after it was precipitated with ethanol, it was dissolved in TE.

[0242] The plasmid hATR5Lva/CVIDEC was digested with the restrictionenzyme KpnI (Takara Shuzo) at 37° C. for 1 hour, and then with therestriction enzyme PstI (Takara Shuzo) at 37° C. for 1 hour. Thedigestion mixture was separated by agarose gel electrophoresis using a1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose stripshaving about 3000 bp long DNA fragments were excised. The agarose stripwas extracted with phenol and chloroform, and after the DNA fragmentswere precipitated with ethanol, they were dissolved in TE.

[0243] The KpnI-PstI DNA fragment prepared as above encoding the FR3 ofversions “b”, or “c” and the hATR5Lva/CVIDEC vector in which the FR3 wasremoved by digesting with KpnI and PstI were ligated using the DNAligation kit ver.2 (Takara Shuzo) by reacting at 16° C. for 1 houraccording to the instructions attached to the kit.

[0244] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of Hi-Competence Broth (Nippongene)was added thereto, incubated at 37° C. for 1 hour, and then the E. coliwas plated on a 100 μg/ml LBA agar medium and incubated overnight at 37°C. to obtain an E. coli transformant. The transformant was culturedovernight in 3 ml of the LBA medium, and from the cell fractions,plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN).

[0245] The nucleotide sequence of the cDNA coding region in the plasmidwas determined using the Dye Terminator Cycle Sequencing FS ReadyReaction Kit (Perkin-Elmer) and the DNA Sequencer 373A (Perkin-Elmer).As the sequencing primer, M13 Primer M4 (Takara Shuzo) and M13 Primer RV(Takara Shuzo) were used, and the sequence was determined by confirmingthe nucleotide sequence in both directions.

[0246] The plasmids that contain the gene encoding version “b” orversion “c” in which the FR3 of humanized antibody L chain version “a”was replaced was designated as hATR5Lvb/CVIDEC or hATR5Lvc/CVIDEC,respectively. The nucleotide sequence and the corresponding amino acidsequence of the humanized L chain version “b” contained in plasmidhATR5Lvb/CVIDEC and the amino acid sequence of version “b” are shown inSEQ ID NO: 87 and 88. The nucleotide sequence and the correspondingamino acid sequence of the humanized L chain version “c” contained inplasmid hATR5Lvc/CVIDEC and the amino acid sequence of version “c” areshown in SEQ ID NO: 89 and 90.

[0247] (iii) Versions “b1” and “b2”

[0248] Versions “b1” and “b2” were generated by replacing the FR2 ofversion “b”. For version “b1” the FR2 derived from human antibody S65921(DDBJ, Tonge D W et al., Year Immunol., 7: 56-62, 1993) was used, andfor version “b2” the FR2 derived from human antibody X93625 (DDBJ, Cox JP et al., Eur. J. Immunol., 24: 827-836, 1994) was used, respectively.

[0249] Primers F2SS (SEQ ID NO: 91) and F2SA (SEQ ID NO: 92) encodingthe FR2 of version “b1”, or primers F2XS (SEQ ID NO: 93) and F2XA (SEQID NO: 94) encoding the FR2 of version “b2” have a sequencecomplementary to each other, and have the recognition sequences of therestriction enzymes AflII and SpeI on both ends thereof. F2SS, F2SA,F2XS, and F2XA were synthesized by Pharmacia Biotech. 100 pmole each ofF2SS and F2SA, or F2XS and F2XA were annealed by treating at 96° C. for2 minutes and at 50° C. for 2 minutes, and the double stranded DNAfragments were generated.

[0250] These double stranded DNA fragments were digested with therestriction enzymes AflII (Takara Shuzo) and SpeI (Takara Shuzo) at 37°C. for 1 hour. The digestion mixture was extracted with phenol andchloroform, and after the DNA fragments were precipitated with ethanol,they were dissolved in TE.

[0251] The plasmid hATR5Lvb/CVIDEC was digested with the restrictionenzymes AflII (Takara Shuzo) and SpeI (Takara Shuzo) at 37° C. for 1hour. The digestion mixture was separated by agarose gel electrophoresisusing a 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarosestrips having about 3000 bp long DNA fragments were excised. The agarosestrip was extracted with phenol and chloroform, and after the DNAfragments were precipitated with ethanol, they were dissolved in TE.

[0252] The AflII-SpeI DNA fragment prepared as above encoding the FR2 ofversion “b1” or “b2” and the hATR5Lvb/CVIDEC vector in which the FR2 wasremoved by digesting with AflII and SpeI were ligated using the DNAligation kit ver.2 (Takara Shuzo) by reacting at 16° C. for 1 houraccording to the instructions attached to the kit.

[0253] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of the Hi-Competence Broth(Nippongene) was added thereto, incubated at 37° C. for 1 hour, and thenthe E. coli was plated on a 100 μg/ml LBA agar medium and incubatedovernight at 37° C. to obtain an E. coli transformant. The transformantwas cultured overnight at 37° C. in 4 ml of the LBA medium and, from thecell fractions, plasmid DNA was prepared using the QIAprep Spin PlasmidKit (QIAGEN).

[0254] The nucleotide sequence of the cDNA coding region in the plasmidwas determined using the Dye Terminator Cycle Sequencing FS ReadyReaction Kit (Perkin-Elmer) by the DNA Sequencer 373A (Perkin-Elmer). Asthe sequencing primer, M13 Primer M4 (Takara Shuzo) and M13 Primer RV(Takara Shuzo) were used, and the sequence was determined by confirmingthe nucleotide sequence in both directions.

[0255] The plasmids that contain the gene encoding version “b1” or “b2”in which the FR2 of humanized antibody L chain version “b” was replacedwas designated as hATR5Lvbl/CVIDEC and hATR5Lv2/CVIDEC, respectively.The nucleotide sequence and the corresponding amino acid sequence of thehumanized L chain version “b1” contained in plasmid hATR5Lvbl/CVIDEC andthe amino acid sequence of version “b1” are shown in SEQ ID NO: 95 and96. The nucleotide sequence and the corresponding amino acid sequence ofthe humanized L chain version “b2” contained in plasmid hATR5Lvb2/CVIDECand the amino acid sequence of version “b2” are shown in SEQ ID NO: 97and 98.

[0256] (3) Construction of the Expression Vector of Humanized Antibody

[0257] (i) Combination of Humanized H Chain ad Chimeric L Chain

[0258] The plasmid hATR5Hva/CVIDEC containing a H chain V region wasdigested with NheI and SalI, and a cDNA fragment of the humanized Hchain V region was recovered and introduced to chATR5/N5KG4P (SalI/NheI)prepared by digesting chATR5/N5KG4P, a chATR-5 antibody expressionplasmid vector, with NheI and SalI. The plasmid thus generated wasdesignated as hHva-chLv/N5KG4P.

[0259] The plasmid hATR5Hvb/CVIDEC containing a H chain V region wasdigested with NheI and SalI, and a cDNA fragment of the humanized Hchain V region was recovered and introduced to chATR5/N5KG4P (SalI/NheI)prepared by digesting chATR5/N5KG4P, a chATR-5 antibody expressionplasmid vector, with NheI and SalI. The plasmid thus generated wasdesignated as hHvb-chLv/N5KG4P.

[0260] The plasmids hATR5Hvc/CVIDEC, hATR5Hvd/CVIDEC, andhATR5Hve/CVIDEC containing a H chain V region were digested with NheIand SalI, and cDNA fragments of the humanized H chain V region wererecovered and introduced to chATR5/N5KG4P (SalI/NheI) prepared bydigesting chATR5/N5KG4P, a chATR-5 antibody expression plasmid vector,with NheI and SalI. The plasmids thus generated were designated ashHvc-chLv/N5KG4P, hHvd-chLv/N5KG4P, and hHve-chLv/N5KG4P.

[0261] The plasmids hATR5Hvf/CVIDEC and hATR5Hvh/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tochATR5/N5KG4P (SalI/NheI) prepared by digesting chATR5/N5KG4P, a chATR-5antibody expression plasmid vector, with NheI and SalI. The plasmidsthus generated were designated as hHvf-chLv/N5KG4P and hHvh-chLv/N5KG4P.

[0262] The plasmids hATR5Hvi/CVIDEC and hATR5Hvj/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tochATR5/N5KG4P (SalI/NheI) prepared by digesting chATR5/N5KG4P, a chATR-5antibody expression plasmid vector, with NheI and SalI. The plasmidsthus generated were designated as hHvi-chLv/N5KG4P and hHvj-chLv/N5KG4P.

[0263] The plasmids hATR5Hb1/CVIDEC and hATR5Hvdl/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tochATR5/N5KG4P (SalI/NheI) prepared by digesting chATR5/N5KG4P, a chATR-5antibody expression plasmid vector, with NheI and SalI. The plasmidsthus generated were designated as hHvbl-chLv/N5KG4P andhHvdl-chLv/N5KG4P.

[0264] (ii) Combination of Humanized L Chain ad Chimeric H Chain

[0265] Using an antibody expression vector N5KG4P, it was combined witha chimeric H chain and was expressed, and the humanized L chain wasevaluated.

[0266] The plasmids hATR5Lva/CVIDEC, hATR5Lvb/CVIDEC, hATR5Lvc/CVIDEC,hATR5Lvbl/CVIDEC, and hATR5Lvb2/CVIDEC were digested with therestriction enzymes BglII (Takara Shuzo) and SplI (Takara Shuzo) at 37°C. for 2-3 hours. The digestion mixture was separated by agarose gelelectrophoresis using a 1.5% or 2% NuSieve GTG agarose (FMCBioProducts), and the agarose strips having about 400 bp long DNAfragments were excised. The agarose strips were extracted with phenoland chloroform, and after the DNA fragments were precipitated withethanol, they were dissolved in TE.

[0267] The SplI-BglII DNA fragment containing the gene encoding the ahumanized L chain V region of each of these versions and thehATR5Hv/N5KG4P digested with SplI and BglII were ligated using the DNAligation kit ver.2 (Takara Shuzo) by reacting at 16° C. for 1 houraccording to the instructions attached to the kit.

[0268] The ligation mixture was added to 100 μl of E. coli JM109competent cells (Nippongene) and was incubated for 30 minutes on ice andfor 1 minute at 42° C. Then, 300 μl of the Hi-Competence Broth(Nippongene) was added thereto, incubated at 37° C. for 1 hour, and thenthe E. coli was plated on a 100 μg/ml LBA agar medium and incubatedovernight at 37° C. to obtain an E. coli transformant.

[0269] The transformant was cultured overnight at 37° C. in 250 ml or500 ml of the LBA medium, and from the cell fractions, plasmid DNA wasprepared using the Plasmid Maxi Kit (QIAGEN). The plasmids in which agene encoding the chimeric H chain and humanized L chain was introducedwere designated as chHv-hLva/N5KG4P, chHv-hLvb/N5KG4P, chHv-hLvc/N5KG4P,chHv-hLvbl/N5KG4P, and chHv-hLvb2/N5KG4P.

[0270] (iii) Combination of Humanized H Chain and Humanized L Chain

[0271] The plasmid hATR5Hva/CVIDEC containing a H chain V region wasdigested with NheI and SalI, and a cDNA fragment of the humanized Hchain V region was recovered and introduced to hLva/N5KG4P (SalI/NheI)prepared by digesting plasmid chHv-hLva/N5KG4P containing the cDNAsequence of humanized ATR-5 antibody L chain version “a” with NheI andSalI. The plasmid thus generated was designated as hHva-hLva/N5KG4P.

[0272] The plasmids hATR5Hvb/CVIDEC and hATR5HvC/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tohLva/N5KG4P (SalI/NheI) prepared by digesting plasmid chHv-hLva/N5KG4Pcontaining the cDNA sequence of humanized ATR-5 antibody L chain version“a” with NheI and SalI. The plasmids thus generated were designated ashHvb-hLva/N5KG4P and hHvc-hLva/N5KG4P.

[0273] The plasmids hATR5Hvb/CVIDEC, hATR5Hvd/CVIDEC, andhATR5Hve/CVIDEC containing a H chain V region were digested with NheIand SalI, and cDNA fragments of the humanized H chain V region wererecovered and introduced to hLvb/N5KG4P (SalI/NheI) prepared bydigesting plasmid chHv-hLvb/N5KG4P containing the cDNA sequence ofhumanized ATR-5 antibody L chain version “b” with NheI and SalI. Theplasmids thus generated were designated as hHvb-hLvb/N5KG4P,hHvd-hLvb/N5KG4P, and hHve-hLvb/N5KG4P.

[0274] The plasmids hATR5Hvf/CVIDEC, hATR5Hvg/CVIDEC, andhATR5Hvh/CVIDEC containing a H chain V region were digested with NheIand SalI, and cDNA fragments of the humanized H chain V region wererecovered and introduced to hLvb/N5KG4P (SalI/NheI) prepared bydigesting plasmid chHv-hLvb/N5KG4P containing the cDNA sequence ofhumanized ATR-5 antibody L chain version “b” with NheI and SalI. Theplasmids thus generated were designated as hHvf-hLvb/N5KG4P,hHvg-hLvb/N5KG4P, and hHvh-hLvb/N5KG4P.

[0275] The plasmids hATR5Hvi/CVIDEC and hATR5Hvj/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tohLvb/N5KG4P (SalI/NheI) prepared by digesting plasmid chHv-hLvb/N5KG4Pcontaining the cDNA sequence of humanized ATR-5 antibody L chain version“b” with NheI and SalI. The plasmids thus generated were designated ashHvi-hLvb/N5KG4P and hHvj-hLvb/N5KG4P.

[0276] The plasmids hATR5Hvbl/CVIDEC and hATR5Hvdl/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tohLvb/N5KG4P (SalI/NheI) prepared by digesting plasmid chHv-hLvb/N5KG4Pcontaining the cDNA sequence of humanized ATR-5 antibody L chain version“b” with NheI and SalI. The plasmids thus generated were designated ashHvb1-hLvb/N5KG4P and hHvdl-hLvb/N5KG4P.

[0277] The plasmids hATR5Hvb3/CVIDEC and hATR5Hvd3/CVIDEC containing a Hchain V region were digested with NheI and SalI, and cDNA fragments ofthe humanized H chain V region were recovered and introduced tohLvb/N5KG4P (SalI/NheI) prepared by digesting plasmid chHv-hLvb/N5KG4Pcontaining the cDNA sequence of humanized ATR-5 antibody L chain version“b” with NheI and SalI. The plasmids thus generated were designated ashHvb3-hLvb/N5KG4P and hHvd3-hLvb/N5KG4P.

[0278] The plasmid hATR5Hvb/CVIDEC containing a H chain V region wasdigested with NheI and SalI, and a cDNA fragment of the humanized Hchain V region was recovered and introduced to hLvbl/N5KG4P (SalI/NheI)and hLvb2/N5KG4P (SalI/NheI) prepared by digesting plasmidschHv-hLvbl/N5KG4P and chHv-hLvb2/N5KG4P containing the cDNA sequence ofhumanized ATR-5 antibody L chain versions “b1” and “b2” with NheI andSalI. The plasmids thus generated were designated as hHvb-hLvbl/N5KG4Pand hHvb-hLvb2/N5KG4P.

[0279] The plasmid hATR5Hvi/CVIDEC containing a H chain V region wasdigested with NheI and SalI, and a cDNA fragment of the humanized Hchain V region was recovered and introduced to hLvbl/N5KG4P (SalI/NheI)and hLvb2/N5KG4P (SalI/NheI) prepared by digesting plasmidschHv-hLvbl/N5KG4P and chHv-hLvb2/N5KG4P containing the cDNA sequence ofhumanized ATR-5 antibody L chain versions “b1” and “b2” with NheI andSalI. The plasmids thus generated were designated as hHvi-hLvb1/N5KG4Pand hHvi-hLvb2/N5KG4P.

[0280] (4) Transfection into COS-7 cells

[0281] In order to evaluate the activity of binding to the antigen andneutralizing activity of humanized antibody, the above antibody wastransiently expressed in COS-7 cells.

[0282] The constructed expression plasmid vector was transduced intoCOS-7 cells by electroporation using the Gene Pulser instrument (BioRad). Fifty μg or 20 μg of the plasmid was added to 0.78 ml of COS-7cells suspended in PBS at a cell concentration of 1×10⁷ cells/ml, whichwas subjected to pulses of 1,500 V and 25 μF capacity.

[0283] After 10 minutes of the recovery period at room temperature, theelectroporated cells were suspended in a DMEM medium (GIBCO) containing5% Ultra low IgG fetal bovine serum (GIBCO), and cultured using a 10 cmculture dish or 15 cm culture dish in a 5% CO₂ incubator. Afterculturing for 24 hours, the culture supernatant was aspirated off, andthen a serum-free medium HBCHO (Irvine Scientific) was added. Afterfurther culturing for 72 hours or 96 hours, the culture supernatant wascollected and centrifuged to remove cell debris.

[0284] (5) Purification of Antibody

[0285] From the culture supernatant of the COS-7 cells, the antibody waspurified using the AffiGel Protein A MAPSII kit (Bio Rad) or therProtein A Sepharose Fast Flow (Pharmacia Biotech). Purification usingthe AffiGel Protein A MAPSII kit was carried out according to theinstructions attached to the kit. Purification using the rprotein ASepharose Fast Flow was carried out as follows:

[0286] One ml of rprotein A Sepharose Fast Flow was filled into a columnand the column was equilibrated by 10 volumes of TBS. The culturesupernatant of COS-7 cells was applied to the equilibrated column, whichwas then washed with 10 volumes of TBS. The adsorbed antibody fractionwas eluted by 13.5 ml of 2.5 mM HCl (pH 3.0). The eluate was neutralizedby adding 1.5 ml of 1 M Tris-HCl (pH 8.0).

[0287] By performing ultrafiltration two or three times for the purifiedantibody fraction using the Centriprep 30 or 100 (amicon), the solventwas replaced to TBS, and was finally concentrated to about 1.5 ml.

Reference Example 6 Antibody Quantitation and Activity Evaluation

[0288] (1) Measurement of Antibody Concentration by ELISA

[0289] ELISA Plates for measurement of antibody concentration wereprepared as follows: Each well of a 96-well ELISA plate (Maxisorp, NUNC)was immobilized by 100 μl of goat anti-human IgGγ antibody (BIO SOURCE)prepared to a concentration of 1 μg/ml in the immobilization buffer (0.1M NaHCO₃. 0.02% NaN₃, pH 9.6) (hereinafter referred to as CB). Afterblocking with 200 μl of the dilution buffer (50 mM Tris-HCl, 1 mM MgCl₂,0.1 M NaCl, 0.05% Tween 20, 0.02% NaN₃. 1% bovine serum albumin (BSA),pH 8.1) (hereinafter referred to as DB), the culture supernatant of theCOS-7 cells in which antibody was expressed or purified antibody wereserially diluted with DB, and then added to each well.

[0290] After incubating at room temperature for 1 hour followed bywashing with the Dulbecco PBS containing 0.05% Tween 20 (hereinafterreferred to as RB), 100 μl of alkaline phosphatase-conjugated goatanti-human IgGy antibody (Biosource) which was diluted 1000-fold with DBwas added. After incubating at room temperature for 1 hour followed bywashing with the RB, Sigma104 (p-nitrophenyl phosphate, SIGMA) dissolvedin the substrate buffer (50 mM NaHCO₃, 10 mM MgCl₂, pH 9.8) to 1 mg/mlwas added, and then the absorbance at 405/655 nm was measured using theMicroplate Reader (Bio Rad). As the standard for the measurement ofconcentration, IgG4κ (Binding Site) was used.

[0291] (2) Measurement of the Activity of Binding to the Antigen

[0292] Cell ELISA plates for measurement of antigen binding wereprepared as follows. Cells used were human bladder carcinoma cells J82(ATCC HTB-1). To 60 wells of a 96-well cell culture plate, 1×10⁵ J82cells were inoculated. This was cultured (RPMI1640 medium containing 10%fetal bovine serum (GIBCO)) for one day in a CO₂ incubator to allow thecells to be attached thereto. After discarding the culture liquid, eachwell was washed twice with 300 μl PBS. 100 μl of PBS containing 4%paraformaldehyde (hereinafter referred to as PFA/PBS) was added to eachwell, and placed on ice for 10 minutes to immobilize the cells.

[0293] PFA/PBS was discarded, and each well was washed twice with 300 μlof PBS, and then blocked with 250 μl of DB. The culture supernatant orpurified antibody was serially diluted with DB, 100 μl of which wasadded to each well. After incubating at room temperature for 2 hoursfollowed by washing with RB, 100 μl of alkaline phosphatase-conjugatedgoat anti-human IgGy antibody (BioSource) diluted 1000-fold with DB wasadded. After incubating for 1 hour followed by washing with RB, thesubstrate solution was added, and then absorbance at 405/655 nm wasmeasured using the Microplate Reader (Bio-Rad).

[0294] (3) Measurement of Neutralizing Activity

[0295] The neutralizing activity of mouse antibody, chimeric antibody,and humanized antibody was measured with the inhibiting activity againstthe Factor Xa-production activity by human placenta-derivedthromboplastin, Thromborel S (Boehringer AG), as an index. Thus, 60 μlof the buffer (TBS containing 5 mM CaCl₂ and 0.1% BSA) was added to 10μl of 1.25 mg/ml Thromborel S and 10 μl of appropriately dilutedantibody, which was then incubated in a 96-well plate at roomtemperature for 1 hour. Ten μl each of 3.245 μg/ml human Factor X(Celsus Laboratories) and 82.5 ng/ml human Factor VIIa (Enzyme Research)were added thereto, and then were incubated at room temperature for 1hour.

[0296] Ten μl of 0.5 M EDTA was added to stop the reaction, to which 50μl of the chromogenic substrate solution was added and the absorbance at405/655 nm was determined using the Microplate Reader (Bio Rad). Afterreacting at room temperature for 1 hour, the absorbance at 405/655 nmwas determined again. The neutralizing activity may be determined bycalculating the residual activity (%) from each change in absorbancewith the hourly absorbance change at no antibody addition as a 100%activity.

[0297] The chromogenic substrate solution was prepared by dissolving theTestzyme chromogenic substrate S-2222 (Chromogenix) according to theattached instructions, diluting 2-fold with purified water and mixingwith a polybrene solution (0.6 mg/ml hexadimethylene bromide, SIGMA) at1:1.

[0298] (4) Evaluation of Activity

[0299] (i) Combination of the Humanized H Chain Version “a” and aChimeric L Chain

[0300] An antibody (a-ch) which is the humanized H chain version “a”combined with a chimeric L chain was generated, and was tested for thebinding activity to the antigen by the cell ELISA. The amount bound tothe antigen was found to be decreased at a high concentration. Theneutralizing activity against the antigen by FXa production-inhibitionwas weak as compared to that of the positive control chimeric antibody(ch-ch). Therefore, it was decided to perform the version-up of thehumanized H chain by FR-shuffling. The chimeric antibody used herein wasthe one that was expressed in COS-7 cells, purified, and evaluated.

[0301] (ii) Combination of the Humanized L Chain Version “a” and aChimeric H Chain

[0302] An antibody (ch-a) which is the humanized L chain version “a”combined with a chimeric H chain was generated, and was tested for thebinding activity to the antigen by the cell ELISA. It was found to havethe binding activity equal to or higher than that of the chimericantibody. On the other hand, the neutralizing activity against theantigen was weak as compared to that of the positive control chimericantibody. Therefore, it was decided to perform the version-up of thehumanized L chain by FR-shuffling. The chimeric antibody used herein wasthe one that was expressed in COS-7 cells, purified, and evaluated.

[0303] (iii) Combination of the Humanized H Chain Version “a” and theHumanized L Chain Version “a”

[0304] An antibody (a-a) which is the humanized H chain version “a”combined with the humanized L chain version “a” was generated, and wastested for the binding activity to the antigen by the cell ELISA. Theamount bound to the antigen was found to be decreased in the highconcentration side. The neutralizing activity against the antigen by FXaproduction-inhibition was weak as compared to that of the positivecontrol chimeric antibody. Therefore, it was decided to perform theversion-up of the humanized H chain and L chain by FR-shuffling. Thechimeric antibody used herein was the one that was expressed in COS-7cells, purified, and evaluated.

[0305] (iv) Combination of the Humanized H Chain Versions “b”, “c”, and“d”, and a Chimeric L Chain

[0306] Antibodies (“b-ch”, “c-ch”, and “d-ch”, respectively) which arethe humanized H chain subjected to version-up by FR-shuffling combinedwith a chimeric L chain were generated, and were tested for the bindingactivity to the antigen by the cell ELISA. “d-ch” exhibited a bindingactivity equal to that of the chimeric antibody, and “b-ch” and “c-ch”exhibited a slightly lower binding activity. On the other hand, theneutralizing activity against the antigen as compared to that ofpositive control chimeric antibody was almost equal in “b-ch”, andslightly weak in “d-ch”. In version “c-ch”, it was significantly weakerthan that of the chimeric antibody. Therefore, the humanized H chainversions “b” and “d” were considered the ones of the humanized H chainto exhibit a high activity.

[0307] (v) Combination of the Humanized H Chain Version “b” and theHumanized L Chain Version “a”

[0308] An antibody (b-a) which is the humanized H chain version “b”subjected to version-up by FR-shuffling combined with the humanized Lchain version “a” was generated, and was tested for the binding activityto the antigen by the cell ELISA. The amount bound to the antigen wasfound to be decreased at a high concentration. On the other hand, theneutralizing activity against the antigen was significantly weak ascompared to that of the positive control chimeric antibody. Therefore,“b-a” and “a-a” were the ones that exhibit a high activity. The chimericantibody used herein was the one that was expressed in COS-7 cells,purified, and evaluated.

[0309] (vi) Combination of the Humanized L Chain Versions “b” and “c”,and a Chimeric H Chain

[0310] Antibodies (“ch-b” and “ch-c”, respectively) which are thehumanized L chain versions “b” and “c” combined with a chimeric H chainwere generated, and both of them were found to have the binding activityto the antigen and the neutralizing activity against the antigen equalto the chimeric antibody. Therefore, versions “b” and “c” were chosen asa candidate for a humanized antibody L chain. Mouse antibody-derivedversion “b” which is one amino acid smaller in the amino acid residuenumber is considered to be superior to version “c” in terms ofantigenicity. The chimeric antibody used herein was the one that wasexpressed in CHO cells DG44, purified, and evaluated. In the evaluationhereinafter the antibody was used as the positive control.

[0311] (vii) Combination of the Humanized H Chain Version “b” and theHumanized L Chain Versions “b” and “c”

[0312] Antibodies (“b-b” and “b-c”, respectively) which are thehumanized H chain version “b” combined with the humanized L chainversions “b” and “c” were generated, and tested for the binding activityto the antigen and the neutralizing activity against the antigen. Bothof them had a slightly lower activity than that of the chimeric antibodyin both the binding activity and the neutralizing activity.

[0313] (viii) Combination of the Humanized H Chain Versions “b” and “d”,and the Humanized L Chain Version “b”

[0314] Antibodies (“b-b” and “d-b”, respectively) which are thehumanized H chain subjected to version-up by FR-shuffling combined withthe humanized L chain version “b” were generated, and were tested forthe binding activity to the antigen by the cell ELISA. “d-b” exhibited abinding activity equal to that of the chimeric antibody, and “b-b”exhibited a slightly lower binding activity at the high concentration.On the other hand, the neutralizing activity against the antigen ascompared to that of the positive control chimeric antibody was slightlylow in “b-b”, and significantly weak in “d-b”. Therefore, it was shownthat “b-b” is a high neutralizing activity version, whereas “d-b” is ahigh binding activity version.

[0315] (ix) Combination of the Humanized H Chain Version “e”, and aChimeric L Chain and the Humanized L Chain Version “b”

[0316] Antibodies (“e-ch” and “e-b”, respectively) which are thehumanized L chain version “e” combined with a chimeric L chain and thehumanized version “b” were generated. “e-ch” exhibited a bindingactivity to the antigen equal to that of the chimeric antibody, but in“e-b” the amount of antibody expressed was very little and most of thebinding activity was lost. The neutralizing activity against the antigenof “e-ch” was significantly low as compared to that of the chimericantibody. Therefore, it was concluded that the H chain version “e”combined with L chain version “b” did not work well.

[0317] (x) Combination of the Humanized H Chain Versions “f”, “g”, and“h”, and the Humanized L Chain Version “b”

[0318] Antibodies (“f-b”, “g-b”, and “h-b”, respectively) which are thehumanized H chain versions “f”, “g”, and “h” combined with the humanizedL chain version “b” were generated. In “f-b” and “h-b” antibody, theamount of antibody expressed was very small. For versions “f” and “h”,antibodies combined with the chimeric L chain were generated, but werenot expressed. “g-b” reached saturation at a low concentration, andexhibited a binding activity weaker than that of the chimeric antibody.The neutralizing activity against the antigen of “g-b” was significantlyweak as compared to that of the chimeric antibody.

[0319] (xi) Combination of the Humanized H Chain Versions “b1” and “d1”,and the Humanized L Chain Version “b”

[0320] Antibodies (“b1-b” and “d1-b”, respectively) which are thehumanized H chain versions “b1” and “d1” combined with the humanized Lchain version “b” were generated. Almost no antibody was expressed inany of them. For these, antibodies combined with a chimeric L chain weregenerated, but were not expressed.

[0321] (xii) Combination of the Humanized H Chain Versions “b3” and“d3”, and the Humanized L Chain Version “b”

[0322] Antibodies (“b3-b” and “d3-b”, respectively) which are thehumanized H chain versions “b3” and “d3” combined with the humanized Lchain version “b” were generated. The binding activity to the antigen of“d3-b” was slightly lower than that of the chimeric antibody, and thatof “b3-b” was much lower. The neutralizing activity against the antigenof “b3-b” was higher than that of “b-b”, but was lower than that of thechimeric antibody, and “d3-b” and “b-b” remained equal in activity.

[0323] (xiii) Combination of the Humanized H Chain Versions “i” and “j”,and a Chimeric L Chain and the Humanized L Chain Version “b”

[0324] Antibodies (“i-ch” and “j-ch”, respectively) which are thehumanized H chain versions “i” and “j” combined with a chimeric L chain,and antibodies (“i-b” and “j-b”, respectively) combined with thehumanized L chain version “b” were generated, and were tested for thebinding activity to the antigen and the neutralizing activity againstthe antigen. The binding activity of any of the antibodies was almostequal to that of the chimeric antibody. “i-ch” exhibited theneutralizing activity higher than that of the chimeric antibody, and“j-ch” was significantly lower than that of the chimeric antibody. “i-b”exhibited the neutralizing activity equal to that of the chimericantibody, and “j-b” exhibited a significantly weaker neutralizingactivity than that of that of the chimeric antibody.

[0325] (xiv) The Humanized L Chain Versions “b1” and “b2”

[0326] When antibodies (“ch-b1” and “ch-b2”, respectively) which are thehumanized L chain versions “b1” and “b2” combined with a chimeric Hchain were generated, both of them exhibited the binding activity to theantigen equal to that of the chimeric antibody. For the neutralizingactivity against the antigen, “ch-b1” exhibited the binding activityequal to that of the chimeric antibody, while “ch-b2” exhibited anactivity slightly higher than that of the chimeric antibody at the highconcentration. Versions “b1” and “b2” can be candidates of a humanizedantibody L chain, but “b2” is superior in that it has a strongeractivity.

[0327] (xv) Combination of the Humanized H Chain Version “b” and theHumanized L Chain Version “b2”

[0328] An antibody (“b-b2”) which is the humanized H chain version “b”combined with the humanized L chain version “b2” was generated, and wastested for the binding activity to the antigen and the neutralizingactivity against the antigen. The binding activity was slightly lowerthan that of the chimeric antibody. The neutralizing activity, thoughslightly higher than that of “b-b”, was lower than that of “i-b”.

[0329] (xvi) Combination of the Humanized H Chain Version “i” and, theHumanized L Chain Version “b1” or “b2”

[0330] Antibodies (“i-b1” and “i-b2”, respectively) which are thehumanized H chain version “i” combined with the humanized L chainversion “b1” or “b2”, were generated, and were tested for the bindingactivity to the antigen and the neutralizing activity against theantigen. The binding activity of “i-b2” was almost equal to that of thechimeric antibody, and that of “i-b1” was slightly lower than that ofchimeric antibody. The neutralizing activity of “i-b1” and “i-b2” washigher than that of the chimeric antibody and “i-b”, which was in adecreasing order of “i-b2”>“i-b1”.

Reference Example 7 Preparation of CHO Cell-Producing Humanized Antibodyand the Evaluation of its Activity

[0331] (1) Establishment of a Cell Line that Stably Produces CHO

[0332] In order to establish cell lines that stably produce a humanizedantibody (b-b, i-b, and i-b2), an antibody expression gene vector wasintroduced into CHO cells (DG44) acclimated to a serum-free medium.

[0333] Plasmid DNA, hHvb-hLvb/N5KG4P, hHvi-hLvb/N5KG4P, andhHvi-hLvb2/N5KG4P were digested with the restriction enzyme SspI (TakaraShuzo) and linearized, which was extracted with phenol and chloroform,and purified by ethanol precipitation. The linearized expression genevector was introduced into the DG44 cells using the electroporationinstrument (Gene Pulser; Bio Rad). The DG44 cells were suspended in PBSat a cell concentration of 1×10⁷ cells/ml, and to about 0.8 ml of thissuspension 10 or 50 μg of the DNA was added, which was subjected topulses of 1,500 V and 25 μF capacity.

[0334] After 10 minutes of the recovery period at room temperature, thetreated cells were suspended in a CHO-S-SFMII medium (GIBCO) containinghypoxanthine/thymidine (GIBCO) (hereinafter referred to as HT), whichwas inoculated on two 96-well plates (Falcon) at 100 μl/well, andcultured in a CO₂ incubator. Eight to nine hours after the start ofculturing, 100 μl/well of the CHO-S-SFMII medium containing HT and 1mg/ml GENETICIN (GIBCO) was added to change to 500 μg/ml of theGENETICIN selection medium, and the cells into which the antibody genehad been introduced were selected. The medium was changed with a freshone once every 3-4 days with ½ the volume. At a time point about 2 weeksafter changing to the selection medium, an aliquot of the culturesupernatant was recovered from the well in which a favorable cell growthwas observed 4-5 days later. The concentration of antibody expressed inthe culture supernatant was measured by the ELISA described above formeasuring antibody concentration, and cells having a high productionyield of antibody were selected.

[0335] (2) Large Scale Purification of Humanized Antibody

[0336] After the DG44 cell lines selected as above that produce thehumanized antibody (“b-b”, “i-b”, and “i-b2”) were cultured for a fewdays in a 500 ml/bottle of the CHO-S-SFMII medium using a 2 liter rollerbottle (CORNING), the culture medium was harvested and a freshCHO-S-SFMII medium was added and cultured again. The culture medium wascentrifuged to remove the cell debris, and filtered with a 0.22 μm or0.45 μm filter. By repeating this, a total of about 2 liters each of theculture supernatant was obtained. From the culture supernatant obtained,antibody was purified by the ConSep LC100 system (Millipore) connectedto the Protein A affinity column (Poros).

[0337] (3) Measurement of Antibody Concentration by ELISA

[0338] ELISA plates for measurement of antibody concentration wereprepared as follows: Each well of a 96-well ELISA plate (Maxisorp, NUNC)was immobilized with 100 μl of goat anti-human IgGy antibody (BioSource)prepared to a concentration of 1 μg/ml with CB. After blocking with 200μl of DB, the culture supernatant of the CHO cells in which antibody hadbeen expressed or the purified antibody was serially diluted with DB,and added to each well.

[0339] After incubating at room temperature for 1 hour and washing withRB, 100 μl of alkaline phosphatase-conjugated goat anti-human IgGyantibody (BioSource) diluted 1000-fold with DB was added. Afterincubating at room temperature for 1 hour and washing with RB, 100 μl ofthe substrate solution was added, and then the absorbance at 405/655 nmwas measured using the Microplate Reader (Bio Rad). As the standard forthe measurement of concentration, human IgG4K (The Binding Site) wasused.

[0340] (4) Measurement of Activity of Binding to the Antigen

[0341] Cell ELISA plates for measurement of antigen binding wereprepared as follows. Cells used were human bladder carcinoma cells J82(ATCC HTB-1), which were inoculated onto a 96-well cell culture plate ata cell count of 1×10⁶ cells. This was cultured (RPMI1640 mediumcontaining 10% fetal bovine serum (GIBCO)) for one day in a CO₂incubator to allow the cells to be attached thereto. After discardingthe culture liquid, each well was washed twice with PBS. 100 μl ofPFA/PBS was added to each well, and placed on ice for 10 minutes toimmobilize the cells.

[0342] PFA/PBS was discarded, and each well was washed twice with 300 μlof PBS and then blocked with 250 μl of DB. Based on the above result ofmeasurement, the purified antibody was serially diluted with DB startingat 10 μg/ml by a factor of 2, 100 μl of which was added to each well.After incubating at room temperature for 2 hours and washing with RB,100 μl of alkaline phosphatase-conjugated goat anti-human IgGγ antibody(BioSource) diluted 1000-fold with DB was added. After incubating atroom temperature for 1 hour and washing with RB, 100 μl of the substratesolution was added, and then absorbance at 405/655 nm was measured usingthe Microplate Reader (Bio-Rad).

[0343] (5) Measurement of Neutralizing Activity Against TF (InhibitingActivity against FXa Production)

[0344] The Factor Xa production-inhibiting activity of humanizedantibody was measured with the inhibiting activity against the Factor Xaproduction activity by the human placenta-derived thromboplastin,Thromborel S (Boehringer AG), as an index. Thus, 60 μl of the buffer(TBS containing 5 mM CaCl₂ and 0.1% BSA) was added to 10 μl of 5 mg/mlThromborel S and 10 μl of the antibody, which was then incubated in a96-well plate at room temperature for 1 hour. The antibody was seriallydiluted with the buffer, starting at 200 μg/ml, by a factor of 5.

[0345] Ten μl each of 3.245 μg/ml human Factor X (Celsus Laboratories)and 82.5 ng/ml human Factor VIIa (Enzyme Research) were added thereto,and were further incubated at room temperature for 45 minutes. Ten μl of0.5 M EDTA was added to stop the reaction. Fifty μl of the chromogenicsubstrate solution was added thereto and the absorbance at 405/655 nmwas determined by the Microplate Reader (Bio Rad). After reacting atroom temperature for 30 minutes, the absorbance at 405/655 nm wasmeasured again. The residual activity (%) was determined from eachchange in absorbance with the absorbance change for 30 minutes at noantibody addition as a 100% activity.

[0346] The chromogenic substrate solution was prepared by dissolving theTestzyme chromogenic substrate S-2222 (Chromogenix) according to theattached instructions, and mixing with a polybrene solution (0.6 mg/mlhexadimethylene bromide, SIGMA) at 1:1.

[0347] (6) Measurement of Neutralizing Activity Against TF (InhibitingActivity against Factor X-Binding)

[0348] The inhibiting activity against Factor X-binding of humanizedantibody was measured using the human placenta-derived thromboplastin,Thromborel S (Boehringer AG), in which a complex of TF and Factor VIIahad previously been formed and the inhibiting activity against FactorX-binding was measured with the Factor Xa production activity of theTF-Factor VIIa complex as an index. Thus, 60 μl of the buffer (TBScontaining 5 mM CaCl₂ and 0.1% BSA) was added to 10 μl of 5 mg/mlThromborel S and 10 μl of 82.5 ng/ml human Factor VIIa (EnzymeResearch), which was preincubated in a 96-well plate at room temperaturefor 1 hour.

[0349] Ten μl of the antibody solution was added thereto, incubated atroom temperature for 5 minutes, and 10 μl of 3.245 μg/ml human Factor X(Celsus Laboratories) was added and was further incubated at roomtemperature for 45 minutes. The antibody was serially diluted with thebuffer starting at 200 μg/ml by a factor of 2. Ten μl of 0.5 M EDTA wasadded to stop the reaction. Fifty μl of the chromogenic substratesolution was added thereto and the absorbance at 405/655 nm wasdetermined by the Microplate Reader (Bio Rad). After reacting at roomtemperature for 30 minutes, the absorbance at 405/655 nm was measuredagain. The residual activity (%) was determined from each change inabsorbance with the absorbance change for 30 minutes at no antibodyaddition as a 100% activity.

[0350] The chromogenic substrate solution was prepared by dissolving theTestzyme chromogenic substrate S-2222 (Chromogenix) according to theattached instructions, and mixing with a polybrene solution (0.6 mg/mlhexadimethylene bromide, SIGMA) at 1:1.

[0351] (7) Measurement of Neutralizing Activity against TF (InhibitingActivity against the Plasma Coagulation)

[0352] The neutralizing activity against TF (inhibiting activity againstthe plasma coagulation) of humanized antibody was measured using, as anindex, the prothrombin time determined using the human placenta-derivedthromboplastin, Thromborel S (Boehringer AG). Thus, 100 μl of humanplasma (Cosmo Bio) was placed into a sample cup, to which 50 μl ofantibody diluted at various concentrations was added, and heated at 37°C. for 3 minutes. Fifty μl of 1.25 mg/ml Thromborel S that hadpreviously been preheated at 37° C. was added to start plasmacoagulation. The coagulation time was measured using the Amelung KC-10Aconnected to the Amelung CR-A (both from M. C. Medical).

[0353] The antibody was serially diluted with TBS containing 0.1% BSA(hereinafter referred to as BSA-TBS) starting at 80 μg/ml by a factor of2. With the coagulation time of no antibody addition as 100% TF plasmacoagulation activity, the residual TF activity was calculated from eachcoagulation time at antibody addition based on a standard curve obtainedby plotting the concentration of Thromborel S and the coagulation time.

[0354] The standard curve was created from the various concentration ofThromborel S and the coagulation time was measured. Fifty μl of BSA-TBSwas added to 50 μl of appropriately diluted Thromborel S, which washeated at 37° C. for 3 minutes, 100 μl of human plasma preheated at 37°C. was added to start coagulation, and the coagulation time wasdetermined. Thromborel S was serially diluted with the Hank's buffer(GIBCO) containing 25 mM CaCl₂ starting at 6.25 mg/ml by a factor of 2.The Thromborel S concentration was plotted on the abscissa, and thecoagulation time on the ordinate on a log-log paper, which was rendereda standard curve.

[0355] (8) Activity Evaluation

[0356] All humanized antibodies, “b-b”, “i-b”, and “i-b2” had anactivity equal to or greater than that of the chimeric antibody (FIG.1). For inhibiting activity against Factor Xa production, inhibitingactivity Factor X-binding, and inhibiting activity against plasmacoagulation as well, the humanized antibodies, “b-b”, “i-b”, and “i-b2”had an activity equal to or greater than that of the chimeric antibody,and the activity was of a decreasing order “i-b2”>“i-b”>“b-b” (FIGS. 2,3, and 4).

1 123 1 28 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer MHC-G1 1 ggatcccggg ccagtggata gacagatg 28 2 27 DNAArtificial Sequence Description of Artificial Sequence Synthetic primerMKC 2 ggatcccggg tggatggtgg gaagatg 27 3 17 DNA Artificial SequenceDescription of Artificial Sequence Synthetic M13 Primer M4 3 gttttcccagtcacgac 17 4 17 DNA Artificial Sequence Description of ArtificialSequence Synthetic M13 Primer RV 4 caggaaacag ctatgac 17 5 408 DNA Musmusculus CDS (1)..(408) sig_peptide (1)..(57) mat_peptide (58)..(408)Nucleotide sequence coding for H chain V region of anti-TF mousemonoclonal antibody ATR-5 5 atg aaa tgc agc tgg gtc atc ttc ttc ctg atggca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met AlaVal Val Thr Gly -15 -10 -5 gtc aat tca gag gtt cag ctg cag cag tct gggact aac ctt gtg agg 96 Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly ThrAsn Leu Val Arg -1 1 5 10 cca ggg gcc tta gtc aag ttg tcc tgc aaa ggttct ggc ttc aac att 144 Pro Gly Ala Leu Val Lys Leu Ser Cys Lys Gly SerGly Phe Asn Ile 15 20 25 aaa gac tac tat atg cac tgg gtg aag cag agg cctgaa cag ggc ctg 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro GluGln Gly Leu 30 35 40 45 gag tgg att gga ggg aat gat cct gcg aat ggt catagt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His SerMet Tyr Asp 50 55 60 ccg aaa ttc cag ggc aag gcc agt ata aca gca gac acatcc tcc aac 288 Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr SerSer Asn 65 70 75 aca gcc tac ctg cag ctc agc agc ctg aca tct gag gac actgcc gtc 336 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr AlaVal 80 85 90 tat ttc tgt gct aga gac tcg ggc tat gct atg gac tac tgg ggtcaa 384 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln95 100 105 gga acc tca gtc acc gtc tcc tca 408 Gly Thr Ser Val Thr ValSer Ser 110 115 6 381 DNA Mus musculus CDS (1)..(381) sig_peptide(1)..(60) mat_peptide (61)..(381) Nucleotide sequence coding for L chainV region of anti-TF mouse monoclonal antibody ATR-5 6 atg agg gcc cctgct cag ttt ttt ggg atc ttg ttg ctc tgg ttt cca 48 Met Arg Ala Pro AlaGln Phe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 ggt atc agatgt gac atc aag atg acc cag tct cca tcc tct atg tat 96 Gly Ile Arg CysAsp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr -1 1 5 10 gca tcg ctggga gag aga gtc act atc act tgc aag gcg agt cag gac 144 Ala Ser Leu GlyGlu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 att aaa agc ttttta agt tgg tac cag caa aaa cca tgg aaa tct cct 192 Ile Lys Ser Phe LeuSer Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro 30 35 40 aag acc ctg atc tattat gca aca agc ttg gca gat ggg gtc cca tca 240 Lys Thr Leu Ile Tyr TyrAla Thr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 aga ttc agt ggc agtgga tct ggg caa gat tat tct cta acc atc aac 288 Arg Phe Ser Gly Ser GlySer Gly Gln Asp Tyr Ser Leu Thr Ile Asn 65 70 75 aac ctg gag tct gac gataca gca act tat tat tgt cta cag cat ggt 336 Asn Leu Glu Ser Asp Asp ThrAla Thr Tyr Tyr Cys Leu Gln His Gly 80 85 90 gag agc ccg tac acg ttc ggaggg ggg acc aag ctg gaa ata aaa 381 Glu Ser Pro Tyr Thr Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 95 100 105 7 35 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer ch5HS 7 gtctgtcgacccaccatgaa atgcagctgg gtcat 35 8 28 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic primer ch5HA 8 tgttgctagc tgaggagacggtgactga 28 9 35 DNA Artificial Sequence Description of ArtificialSequence Synthetic primer ch5LS 9 gtctagatct ccaccatgag ggcccctgct cagtt35 10 28 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer ch5LA 10 tgttcgtacg ttttatttcc agcttggt 28 11 104 DNAArtificial Sequence Description of Artificial Sequence Synthetic CDRgrafting primer hR5Hv1S 11 ttctgtcgac ccaccatgaa atgcagctgg gtcatcttcttcctgatggc agtggttaca 60 ggggttaact cacaggtgca gctgttggag tctggagctgtgct 104 12 108 DNA Artificial Sequence Description of ArtificialSequence Synthetic CDR grafting primer hR5Hv28 12 acaggtgcag ctgttggagtctggagctgt gctggcaagg cctgggactt ccgtgaagat 60 ctcctgcaag gcttccggattcaacattaa agactactat atgcattg 108 13 108 DNA Artificial SequenceDescription of Artificial Sequence Synthetic CDR grafting primer hR5Hv4S13 gaatggccat agtatgtatg acccgaaatt ccagggcagg gccaaactga ctgcagccac 60atccgccagt attgcctact tggagttctc gagcctgaca aatgagga 108 14 110 DNAArtificial Sequence Description of Artificial Sequence Synthetic CDRgrafting primer hR5Hv3A 14 tcatacatac tatggccatt cgcaggatca ttcccaccaatccattctag accctgtcca 60 ggcctctgtt ttacccaatg catatagtag tctttaatgttgaatccgga 110 15 110 DNA Artificial Sequence Description of ArtificialSequence Synthetic CDR grafting primer hR5Hv5A 15 agaagctagc tgaggagacggtgaccaggg tgccttggcc ccagtagtcc atggcatagc 60 ccgagtctct tgcacagtaatagaccgcag aatcctcatt tgtcaggctc 110 16 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer hR5HvPrS 16ttctgtcgac ccaccatga 19 17 19 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer hR5HvPrA 17 agaagctagc tgaggagac 1918 414 DNA Artificial Sequence Description of Artificial SequenceSynthetic nucleotide sequence coding for version “a” of humanized Hchain V region 18 atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtggtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val ValThr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tct gga gct gtgctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val LeuAla Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aag gct tcc ggattc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly PheAsn Ile 15 20 25 aaa gac tac tat atg cat tgg gta aaa cag agg cct gga cagggt cta 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu 30 35 40 45 gaa tgg att ggt ggg aat gat cct gcg aat ggc cat agt atgtat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met TyrAsp 50 55 60 ccg aaa ttc cag ggc agg gcc aaa ctg act gca gcc aca tcc gccagt 288 Pro Lys Phe Gln Gly Arg Ala Lys Leu Thr Ala Ala Thr Ser Ala Ser65 70 75 att gcc tac ttg gag ttc tcg agc ctg aca aat gag gat tct gcg gtc336 Ile Ala Tyr Leu Glu Phe Ser Ser Leu Thr Asn Glu Asp Ser Ala Val 8085 90 tat tac tgt gca aga gac tcg ggc tat gcc atg gac tac tgg ggc caa384 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr Leu Val ThrVal Ser Ser Ala Ser 110 115 19 119 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic amino acid sequence of version “a” ofhumanized H chain V region 19 Gln Val Gln Leu Leu Glu Ser Gly Ala ValLeu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala SerGly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg ProGly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Lys Leu Thr Ala AlaThr Ser Ala Ser Ile Ala Tyr 65 70 75 80 Leu Glu Phe Ser Ser Leu Thr AsnGlu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Tyr Ala MetAsp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser115 20 100 DNA Artificial Sequence Description of Artificial SequenceSynthetic FR Shuffling primer F3RFFS 20 ttcttggcca tagtatgtat gacccgaaattccagggccg agtcacaatc actgcagaca 60 catccacgaa cacagcctac atggagctctcgagtctgag 100 21 75 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3RFBS 21 ggagctctcg agtctgagatctgaggacac agccatttat tactgtgcaa gagactcggg 60 ctatgccatg gttct 75 22100 DNA Artificial Sequence Description of Artificial Sequence SyntheticFR Shuffling primer F3RFFA 22 ctcagactcg agagctccat gtaggctgtgttcgtggatg tgtctgcagt gattgtgact 60 cggccctgga atttcgggtc atacatactatggccaagaa 100 23 75 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3RFBA 23 agaaccatgg catagcccgagtctcttgca cagtaataaa tggctgtgtc ctcagatctc 60 agactcgaga gctcc 75 24100 DNA Artificial Sequence Description of Artificial Sequence SyntheticFR Shuffling primer F3NMFS 24 ttcttggcca tagtatgtat gacccgaaattccagggccg agtcacaatg ctggtagaca 60 catccaagaa ccagttctcc ctgaggctctcgagtgtgac 100 25 75 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3NMBS 25 gaggctctcg agtgtgacagccgcggacac agccgtatat tactgtgcaa gagactcggg 60 ctatgccatg gttct 75 26100 DNA Artificial Sequence Description of Artificial Sequence SyntheticFR Shuffling primer F3NMFA 26 gtcacactcg agagcctcag ggagaactggttcttggatg tgtctaccag cattgtgact 60 cggccctgga atttcgggtc atacatactatggccaagaa 100 27 75 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3NMBA 27 agaaccatgg catagcccgagtctcttgca cagtaatata cggctgtgtc cgcggctgtc 60 acactcgaga gcctc 75 28414 DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence coding for version “b” of humanized H chain V region28 atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10-5 gtt aac tca cag gtg cag ctg ttg gag tct gga gct gtg ctg gca agg 96Val Asn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 510 cct ggg act tcc gtg aag atc tcc tgc aag gct tcc gga ttc aac att 144Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25aaa gac tac tat atg cat tgg gta aaa cag agg cct gga cag ggt cta 192 LysAsp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45gaa tgg att ggt ggg aat gat cct gcg aat ggc cat agt atg tat gac 240 GluTrp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ccgaaa ttc cag ggc cga gtc aca atc act gca gac aca tcc acg aac 288 Pro LysPhe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn 65 70 75 aca gcctac atg gag ctc tcg agt ctg aga tct gag gac aca gcc att 336 Thr Ala TyrMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile 80 85 90 tat tac tgtgca aga gac tcg ggc tat gcc atg gac tac tgg ggc caa 384 Tyr Tyr Cys AlaArg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 ggc acc ctggtc acc gtc tcc tca gct agc 414 Gly Thr Leu Val Thr Val Ser Ser Ala Ser110 115 29 119 PRT Artificial Sequence Description of ArtificialSequence Synthetic amino acid sequence of version “b” of humanized Hchain V region 29 Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala ArgPro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe AsnIle Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met TyrAsp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser ThrAsn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Ile Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser 115 30 414DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence coding for version“c” of humanized H chain V region30 atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10-5 gtt aac tca cag gtg cag ctg ttg gag tct gga gct gtg ctg gca agg 96Val Asn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 510 cct ggg act tcc gtg aag atc tcc tgc aag gct tcc gga ttc aac att 144Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25aaa gac tac tat atg cat tgg gta aaa cag agg cct gga cag ggt cta 192 LysAsp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45gaa tgg att ggt ggg aat gat cct gcg aat ggc cat agt atg tat gac 240 GluTrp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ccgaaa ttc cag ggc cga gtc aca atg ctg gta gac aca tcc aag aac 288 Pro LysPhe Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn 65 70 75 cag ttctcc ctg agg ctc tcg agt gtg aca gcc gcg gac aca gcc gta 336 Gln Phe SerLeu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 80 85 90 tat tac tgtgca aga gac tcg ggc tat gcc atg gac tac tgg ggc caa 384 Tyr Tyr Cys AlaArg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 ggc acc ctggtc acc gtc tcc tca gct agc 414 Gly Thr Leu Val Thr Val Ser Ser Ala Ser110 115 31 119 PRT Artificial Sequence Description of ArtificialSequence Synthetic amino acid sequence of version “c” of humanized Hchain V region 31 Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala ArgPro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe AsnIle Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met TyrAsp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser LysAsn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser 115 32 100DNA Artificial Sequence Description of Artificial Sequence Synthetic FRShuffling primer F3EPS 32 ttcttggcca tagtatgtat gacccgaaat tccagggcagagtcacgatt actgcggacg 60 aatccacgag cacagcctac atggagctct cgagtctgag 10033 75 DNA Artificial Sequence Description of Artificial SequenceSynthetic FR Shuffling primer F3EPA 33 agaaccatgg catagcccga gtctctcgcacagaaatata cggccgagtc ctcagatctc 60 agactcgaga gctcc 75 34 20 DNAArtificial Sequence Description of Artificial Sequence Synthetic primerF3PrS 34 ttcttggcca tagtatgtat 20 35 18 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer F3PrA 35 agaaccatggcatagccc 18 36 100 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3VHS 36 ttcttggcca tagtatgtatgacccgaaat tccagggcag agtctcgatt accgcggacg 60 agtcaacgaa gatagcctacatggagctca acagtctgag 100 37 75 DNA Artificial Sequence Description ofArtificial Sequence Synthetic FR Shuffling primer F3vHA 37 agaaccatggcatagcccga gtctctcgca cagaaataaa cggccgtgtc ctcagatctc 60 agactgttgagctcc 75 38 414 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence coding for version “d” ofhumanized H chain V region 38 atg aaa tgc agc tgg gtc atc ttc ttc ctgatg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu MetAla Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tctgga gct gtg ctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser GlyAla Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aaggct tcc gga ttc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys AlaSer Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cat tgg gta aaa cag aggcct gga cag ggt cta 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg ProGly Gln Gly Leu 30 35 40 45 gaa tgg att ggt ggg aat gat cct gcg aat ggccat agt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc aga gtc acg att act gcg gacgaa tcc acg agc 288 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp GluSer Thr Ser 65 70 75 aca gcc tac atg gag ctc tcg agt ctg aga tct gag gactcg gcc gta 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp SerAla Val 80 85 90 tat ttc tgt gcg aga gac tcg ggc tat gcc atg gac tac tggggc caa 384 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp GlyGln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr LeuVal Thr Val Ser Ser Ala Ser 110 115 39 119 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “d” of humanized H chain 39 Gln Val Gln Leu Leu Glu Ser Gly AlaVal Leu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys AlaSer Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln ArgPro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn GlyHis Ser Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr AlaAsp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu ArgSer Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Ser Gly Tyr AlaMet Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser AlaSer 115 40 414 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence coding for version “e” ofhumanized H chain V region 40 atg aaa tgc agc tgg gtc atc ttc ttc ctgatg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu MetAla Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tctgga gct gtg ctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser GlyAla Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aaggct tcc gga ttc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys AlaSer Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cat tgg gta aaa cag aggcct gga cag ggt cta 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg ProGly Gln Gly Leu 30 35 40 45 gaa tgg att ggt ggg aat gat cct gcg aat ggccat agt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc aga gtc tcg att acc gcg gacgag tca acg aag 288 Pro Lys Phe Gln Gly Arg Val Ser Ile Thr Ala Asp GluSer Thr Lys 65 70 75 ata gcc tac atg gag ctc aac agt ctg aga tct gag gacacg gcc gtt 336 Ile Ala Tyr Met Glu Leu Asn Ser Leu Arg Ser Glu Asp ThrAla Val 80 85 90 tat ttc tgt gcg aga gac tcg ggc tat gcc atg gac tac tggggc caa 384 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp GlyGln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr LeuVal Thr Val Ser Ser Ala Ser 110 115 41 119 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “e” of humanized H chain V region 41 Gln Val Gln Leu Leu Glu SerGly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser CysLys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val LysGln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro AlaAsn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Ser IleThr Ala Asp Glu Ser Thr Lys Ile Ala Tyr 65 70 75 80 Met Glu Leu Asn SerLeu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Ser GlyTyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val SerSer Ala Ser 115 42 100 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3SSS 42 ttcttggcca tagtatgtatgacccgaaat tccagggcag agtcacgatt accgcggaca 60 catccacgag cacagcctacatggagctca ggagcctgag 100 43 75 DNA Artificial Sequence Description ofArtificial Sequence Synthetic FR Shuffling primer F3SSA 43 agaaccatggcatagcccga gtctctcgca cagtaataca cggccgtgtc gtcagatctc 60 aggctcctgagctcc 75 44 100 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3CDS 44 ttcttggcca tagtatgtatgacccgaaat tccagggcaa agccactctg actgcagacg 60 aatcctccag cacagcctacatgcaactct cgagcctacg 100 45 75 DNA Artificial Sequence Description ofArtificial Sequence Synthetic FR Shuffling primer F3CDA 45 agaaccatggcatagcccga gtctcttgca caagaataga ccgcagagtc ctcagatcgt 60 aggctcgagagttgc 75 46 414 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence coding for version “f” ofhumanized H chain V region 46 atg aaa tgc agc tgg gtc atc ttc ttc ctgatg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu MetAla Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tctgga gct gtg ctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser GlyAla Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aaggct tcc gga ttc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys AlaSer Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cat tgg gta aaa cag aggcct gga cag ggt cta 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg ProGly Gln Gly Leu 30 35 40 45 gaa tgg att ggt ggg aat gat cct gcg aat ggccat agt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc aga gtc acg att acc gcg gacaca tcc acg agc 288 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp ThrSer Thr Ser 65 70 75 aca gcc tac atg gag ctc agg agc ctg aga tct gac gacacg gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp ThrAla Val 80 85 90 tat tac tgt gcg aga gac tcg ggc tat gcc atg gac tac tggggc caa 384 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp GlyGln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr LeuVal Thr Val Ser Ser Ala Ser 110 115 47 119 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “f” of humanized H chain V region 47 Gln Val Gln Leu Leu Glu SerGly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser CysLys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val LysGln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro AlaAsn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr IleThr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg SerLeu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser GlyTyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val SerSer Ala Ser 115 48 414 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence coding for version “g” ofhumanized H chain V region 48 atg aaa tgc agc tgg gtc atc ttc ttc ctgatg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu MetAla Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tctgga gct gtg ctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser GlyAla Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aaggct tcc gga ttc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys AlaSer Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cat tgg gta aaa cag aggcct gga cag ggt cta 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg ProGly Gln Gly Leu 30 35 40 45 gaa tgg att ggt ggg aat gat cct gcg aat ggccat agt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc aaa gcc act ctg act gca gacgaa tcc tcc agc 288 Pro Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp GluSer Ser Ser 65 70 75 aca gcc tac atg caa ctc tcg agc cta cga tct gag gactct gcg gtc 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp SerAla Val 80 85 90 tat tct tgt gca aga gac tcg ggc tat gcc atg gac tac tggggc caa 384 Tyr Ser Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp GlyGln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr LeuVal Thr Val Ser Ser Ala Ser 110 115 49 119 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “g” of humanized H chain V region 49 Gln Val Gln Leu Leu Glu SerGly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser CysLys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val LysGln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro AlaAsn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr LeuThr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser SerLeu Arg Ser Glu Asp Ser Ala Val Tyr Ser Cys 85 90 95 Ala Arg Asp Ser GlyTyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val SerSer Ala Ser 115 50 100 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3ADS 50 ttcttggcca tagtatgtatgacccgaaat tccagggccg cgtcaccatg tcagccgaca 60 agtcctccag cgccgcctatttacagtgga ccagccttaa 100 51 75 DNA Artificial Sequence Description ofArtificial Sequence Synthetic FR Shuffling primer F3ADA 51 agaaccatggcatagcccga gtctctcgcg cagaaatata tggcggtgtc cgaggcctta 60 aggctggtccactgt 75 52 414 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence coding for version “h” ofhumanized H chain 52 atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtggtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val ValThr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tct gga gct gtgctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val LeuAla Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aag gct tcc ggattc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly PheAsn Ile 15 20 25 aaa gac tac tat atg cat tgg gta aaa cag agg cct gga cagggt cta 192 Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu 30 35 40 45 gaa tgg att ggt ggg aat gat cct gcg aat ggc cat agt atgtat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met TyrAsp 50 55 60 ccg aaa ttc cag ggc cgc gtc acc atg tca gcc gac aag tcc tccagc 288 Pro Lys Phe Gln Gly Arg Val Thr Met Ser Ala Asp Lys Ser Ser Ser65 70 75 gcc gcc tat tta cag tgg acc agc ctt aag gcc tcg gac acc gcc ata336 Ala Ala Tyr Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile 8085 90 tat ttc tgc gcg aga gac tcg ggc tat gcc atg gac tac tgg ggc caa384 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr Leu Val ThrVal Ser Ser Ala Ser 110 115 53 119 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic amino acid sequence of version “h” ofhumanized H chain V region 53 Gln Val Gln Leu Leu Glu Ser Gly Ala ValLeu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala SerGly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg ProGly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Ser Ala AspLys Ser Ser Ser Ala Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys AlaSer Asp Thr Ala Ile Tyr Phe Cys 85 90 95 Ala Arg Asp Ser Gly Tyr Ala MetAsp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser115 54 100 DNA Artificial Sequence Description of Artificial SequenceSynthetic FR Shuffling primer F3MMS 54 ttcttggcca tagtatgtat gacccgaaattccagggcag agtcacgatt accgcggaca 60 catcgacgag cacagtcttc atggaactgagcagcctgag 100 55 75 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3MMA 55 agaaccatgg catagcccgagtctctcgca cagtaataca cggccgtgtc ttcagatctc 60 aggctgctca gttcc 75 56100 DNA Artificial Sequence Description of Artificial Sequence SyntheticFR Shuffling primer F3BMS 56 ttcttggcca tagtatgtat gacccgaaat tccagggcagagtcaccttt accgcggaca 60 catccgcgaa cacagcctac atggagttga ggagcctcag 10057 75 DNA Artificial Sequence Description of Artificial SequenceSynthetic FR Shuffling primer F3BMA 57 agaaccatgg catagcccga gtctctcgcacaataataaa cagccgtgtc tgcagatctg 60 aggctcctca actcc 75 58 414 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence coding for version “i” of humanized H chain V region58 atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10-5 gtt aac tca cag gtg cag ctg ttg gag tct gga gct gtg ctg gca agg 96Val Asn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 510 cct ggg act tcc gtg aag atc tcc tgc aag gct tcc gga ttc aac att 144Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25aaa gac tac tat atg cat tgg gta aaa cag agg cct gga cag ggt cta 192 LysAsp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45gaa tgg att ggt ggg aat gat cct gcg aat ggc cat agt atg tat gac 240 GluTrp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ccgaaa ttc cag ggc aga gtc acg att acc gcg gac aca tcg acg agc 288 Pro LysPhe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 65 70 75 aca gtcttc atg gaa ctg agc agc ctg aga tct gaa gac acg gcc gtg 336 Thr Val PheMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgtgcg aga gac tcg ggc tat gcc atg gac tac tgg ggc caa 384 Tyr Tyr Cys AlaArg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 ggc acc ctggtc acc gtc tcc tca gct agc 414 Gly Thr Leu Val Thr Val Ser Ser Ala Ser110 115 59 119 PRT Artificial Sequence Description of ArtificialSequence Synthetic amino acid sequence of version “i” of humanized Hchain V region 59 Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala ArgPro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe AsnIle Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met TyrAsp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser ThrSer Thr Val Phe 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser 115 60 414DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence coding for version “j” of humanized H chain V region60 atg aaa tgc agc tgg gtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10-5 gtt aac tca cag gtg cag ctg ttg gag tct gga gct gtg ctg gca agg 96Val Asn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 510 cct ggg act tcc gtg aag atc tcc tgc aag gct tcc gga ttc aac att 144Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25aaa gac tac tat atg cat tgg gta aaa cag agg cct gga cag ggt cta 192 LysAsp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45gaa tgg att ggt ggg aat gat cct gcg aat ggc cat agt atg tat gac 240 GluTrp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ccgaaa ttc cag ggc aga gtc acc ttt acc gcg gac aca tcc gcg aac 288 Pro LysPhe Gln Gly Arg Val Thr Phe Thr Ala Asp Thr Ser Ala Asn 65 70 75 aca gcctac atg gag ttg agg agc ctc aga tct gca gac acg gct gtt 336 Thr Ala TyrMet Glu Leu Arg Ser Leu Arg Ser Ala Asp Thr Ala Val 80 85 90 tat tat tgtgcg aga gac tcg ggc tat gcc atg gac tac tgg ggc caa 384 Tyr Tyr Cys AlaArg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 ggc acc ctggtc acc gtc tcc tca gct agc 414 Gly Thr Leu Val Thr Val Ser Ser Ala Ser110 115 61 119 PRT Artificial Sequence Description of ArtificialSequence Synthetic amino acid sequence of version “j” of humanized Hchain V region 61 Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala ArgPro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe AsnIle Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile 35 40 45 Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met TyrAsp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Phe Thr Ala Asp Thr Ser AlaAsn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Ala Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser 115 62 79DNA Artificial Sequence Description of Artificial Sequence Synthetic FRShuffling primer F2MPS 62 ttctatgcat tgggtgcgcc aggctccagg acagggcctggagtggatgg gagggaatga 60 tcctgcgaat ggccattct 79 63 79 DNA ArtificialSequence Description of Artificial Sequence Synthetic FR Shufflingprimer F2MPA 63 agaatggcca ttcgcaggat cattccctcc catccactcc aggccctgtcctggagcctg 60 gcgcacccaa tgcatagaa 79 64 414 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide sequence codingfor version “b1” of humanized H chain V region 64 atg aaa tgc agc tgggtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp ValIle Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtgcag ctg ttg gag tct gga gct gtg ctg gca agg 96 Val Asn Ser Gln Val GlnLeu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtgaag atc tcc tgc aag gct tcc gga ttc aac att 144 Pro Gly Thr Ser Val LysIle Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cattgg gtg cgc cag gct cca gga cag ggc ctg 192 Lys Asp Tyr Tyr Met His TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga ggg aatgat cct gcg aat ggc cat agt atg tat gac 240 Glu Trp Met Gly Gly Asn AspPro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc cga gtcaca atc act gca gac aca tcc acg aac 288 Pro Lys Phe Gln Gly Arg Val ThrIle Thr Ala Asp Thr Ser Thr Asn 65 70 75 aca gcc tac atg gag ctc tcg agtctg aga tct gag gac aca gcc att 336 Thr Ala Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Ile 80 85 90 tat tac tgt gca aga gac tcg ggc tatgcc atg gac tac tgg ggc caa 384 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr AlaMet Asp Tyr Trp Gly Gln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gctagc 414 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 65 119 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “b1” of humanized H chain V region 65 Gln ValGln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 SerVal Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 TyrMet His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 GlyGly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 GlnGly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser 115 66 414 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide sequence codingfor version “d1” of humanized H chain V region 66 atg aaa tgc agc tgggtc atc ttc ttc ctg atg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp ValIle Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtgcag ctg ttg gag tct gga gct gtg ctg gca agg 96 Val Asn Ser Gln Val GlnLeu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtgaag atc tcc tgc aag gct tcc gga ttc aac att 144 Pro Gly Thr Ser Val LysIle Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cattgg gtg cgc cag gct cca gga cag ggc ctg 192 Lys Asp Tyr Tyr Met His TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga ggg aatgat cct gcg aat ggc cat agt atg tat gac 240 Glu Trp Met Gly Gly Asn AspPro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc aga gtcacg att act gcg gac gaa tcc acg agc 288 Pro Lys Phe Gln Gly Arg Val ThrIle Thr Ala Asp Glu Ser Thr Ser 65 70 75 aca gcc tac atg gag ctc tcg agtctg aga tct gag gac tcg gcc gta 336 Thr Ala Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Ser Ala Val 80 85 90 tat ttc tgt gcg aga gac tcg ggc tatgcc atg gac tac tgg ggc caa 384 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr AlaMet Asp Tyr Trp Gly Gln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gctagc 414 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 67 119 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “d1” of humanized H chain V region 67 Gln ValGln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 SerVal Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 TyrMet His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 GlyGly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 GlnGly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser 115 68 79 DNA Artificial SequenceDescription of Artificial Sequence Synthetic FR Shuffling primer F2VHS68 ttctatgcat tgggtgcgac aggcccctgg acaagggctt gagtggattg gagggaatga 60tcctgcgaat ggccatctt 79 69 79 DNA Artificial Sequence Description ofArtificial Sequence Synthetic FR Shuffling primer F2VHA 69 aagatggccattcgcaggat cattccctcc aatccactca agcccttgtc caggggcctg 60 tcgcacccaatgcatagaa 79 70 414 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence coding for version “b3” ofhumanized H chain V region 70 atg aaa tgc agc tgg gtc atc ttc ttc ctgatg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe Phe Leu MetAla Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttg gag tctgga gct gtg ctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu Glu Ser GlyAla Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcc tgc aaggct tcc gga ttc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser Cys Lys AlaSer Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cat tgg gtg cga cag gcccct gga caa ggg ctt 192 Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 gag tgg att gga ggg aat gat cct gcg aat ggccat agt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly HisSer Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc cga gtc aca atc act gca gacaca tcc acg aac 288 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp ThrSer Thr Asn 65 70 75 aca gcc tac atg gag ctc tcg agt ctg aga tct gag gacaca gcc att 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Ile 80 85 90 tat tac tgt gca aga gac tcg ggc tat gcc atg gac tac tggggc caa 384 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp GlyGln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 Gly Thr LeuVal Thr Val Ser Ser Ala Ser 110 115 71 119 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “b3” of humanized H chain V region 71 Gln Val Gln Leu Leu GluSer Gly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile SerCys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp ValArg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Asn Asp ProAla Asn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val ThrIle Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu SerSer Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Asp SerGly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr ValSer Ser Ala Ser 115 72 414 DNA Artificial Sequence Description ofArtificial Sequence Synthetic nucleotide sequence coding for version“d3” of humanized H chain V region 72 atg aaa tgc agc tgg gtc atc ttcttc ctg atg gca gtg gtt aca ggg 48 Met Lys Cys Ser Trp Val Ile Phe PheLeu Met Ala Val Val Thr Gly -15 -10 -5 gtt aac tca cag gtg cag ctg ttggag tct gga gct gtg ctg gca agg 96 Val Asn Ser Gln Val Gln Leu Leu GluSer Gly Ala Val Leu Ala Arg -1 1 5 10 cct ggg act tcc gtg aag atc tcctgc aag gct tcc gga ttc aac att 144 Pro Gly Thr Ser Val Lys Ile Ser CysLys Ala Ser Gly Phe Asn Ile 15 20 25 aaa gac tac tat atg cat tgg gtg cgacag gcc cct gga caa ggg ctt 192 Lys Asp Tyr Tyr Met His Trp Val Arg GlnAla Pro Gly Gln Gly Leu 30 35 40 45 gag tgg att gga ggg aat gat cct gcgaat ggc cat agt atg tat gac 240 Glu Trp Ile Gly Gly Asn Asp Pro Ala AsnGly His Ser Met Tyr Asp 50 55 60 ccg aaa ttc cag ggc aga gtc acg att actgcg gac gaa tcc acg agc 288 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr AlaAsp Glu Ser Thr Ser 65 70 75 aca gcc tac atg gag ctc tcg agt ctg aga tctgag gac tcg gcc gta 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser GluAsp Ser Ala Val 80 85 90 tat ttc tgt gcg aga gac tcg ggc tat gcc atg gactac tgg ggc caa 384 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp TyrTrp Gly Gln 95 100 105 ggc acc ctg gtc acc gtc tcc tca gct agc 414 GlyThr Leu Val Thr Val Ser Ser Ala Ser 110 115 73 119 PRT ArtificialSequence Description of Artificial Sequence Synthetic amino acidsequence of version “d3” of humanized H chain V region 73 Gln Val GlnLeu Leu Glu Ser Gly Ala Val Leu Ala Arg Pro Gly Thr 1 5 10 15 Ser ValLys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr MetHis Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly GlyAsn Asp Pro Ala Asn Gly His Ser Met Tyr Asp Pro Lys Phe 50 55 60 Gln GlyArg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 MetGlu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 AlaArg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser 115 74 98 DNA Artificial SequenceDescription of Artificial Sequence Synthetic FR Shuffling vector LvlS 74gtctagatct ccaccatgag ggcccctgct cagttttttg ggatcttgtt gctctggttt 60ccagggatcc gatgtgacat ccagatgacc cagtctcc 98 75 98 DNA ArtificialSequence Description of Artificial Sequence Synthetic FR Shufflingvector h5Lv4S 75 ttggcagatg gggtcccatc aaggttcagt ggctccggat ctggtaccgatttcactctc 60 accatctcga gtctgcaacc tgaagatttt gcaactta 98 76 98 DNAArtificial Sequence Description of Artificial Sequence Synthetic FRShuffling vector h5Lv2A 76 cttaagaagc ttttaatgtc ctgtgaggcc ttgcacgtgatggtgactct gtctcctaca 60 gatgcagaca gggaggatgg agactgggtc atctggat 98 7798 DNA Artificial Sequence Description of Artificial Sequence SyntheticFR Shuffling vector h5Lv3A 77 gatgggaccc catctgccaa actagttgcataatagatca ggagcttagg ggctttccct 60 ggtttctgct gataccaact taagaagcttttaatgtc 98 78 94 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling vector h5Lv5A 78 tgttcgtacg tttgatctccaccttggtcc ctccgccgaa cgtgtacggg ctctcaccat 60 gctgcagaca gtagtaagttgcaaaatctt cagg 94 79 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer h5LvS 79 gtctagatct ccaccatgag 2080 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer h5LvA 80 tgttcgtacg tttgatctc 19 81 381 DNA ArtificialSequence Description of Artificial Sequence Synthetic nucleotidesequence coding for version “a” of humanized L chain V region 81 atg agggcc cct gct cag ttt ttt ggg atc ttg ttg ctc tgg ttt cca 48 Met Arg AlaPro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 gggatc cga tgt gac atc cag atg acc cag tct cca tcc tcc ctg tct 96 Gly IleArg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 1 5 10 gcatct gta gga gac aga gtc acc atc acg tgc aag gcc tca cag gac 144 Ala SerVal Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 att aaaagc ttc tta agt tgg tat cag cag aaa cca ggg aaa gcc cct 192 Ile Lys SerPhe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 30 35 40 aag ctc ctgatc tat tat gca act agt ttg gca gat ggg gtc cca tca 240 Lys Leu Leu IleTyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 agg ttc agtggc tcc gga tct ggt acc gat ttc act ctc acc atc tcg 288 Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 agt ctg caa cctgaa gat ttt gca act tac tac tgt ctg cag cat ggt 336 Ser Leu Gln Pro GluAsp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly 80 85 90 gag agc ccg tac acgttc ggc gga ggg acc aag gtg gag atc aaa 381 Glu Ser Pro Tyr Thr Phe GlyGly Gly Thr Lys Val Glu Ile Lys 95 100 105 82 107 PRT ArtificialSequence Description of Artificial Sequence Synthetic amino acidsequence of version “a” of humanized L chain V region 82 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 20 25 30 Leu Ser TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr AlaThr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu AspPhe Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Tyr 85 90 95 Thr PheGly Gly Gly Thr Lys Val Glu Ile Lys 100 105 83 77 DNA ArtificialSequence Description of Artificial Sequence Synthetic FR Shufflingprimer F3SS 83 gtctggtacc gattacactc tcaccatctc gagcctccag cctgaagattttgcaactta 60 ctattgtctg cagaaca 77 84 77 DNA Artificial SequenceDescription of Artificial Sequence Synthetic FR Shuffling primer F3SA 84tgttctgcag acaatagtaa gttgcaaaat cttcaggctg gaggctcgag atggtgagag 60tgtaatcggt accagac 77 85 77 DNA Artificial Sequence Description ofArtificial Sequence Synthetic FR Shuffling primer F3RS 85 gtctggtaccgattacactc tcaccatctc gagcctccag cctgaagata ttgcaactta 60 ctattgtctgcagaaca 77 86 77 DNA Artificial Sequence Description of ArtificialSequence Synthetic FR Shuffling primer F3RA 86 tgttctgcag acaatagtaagttgcaatat cttcaggctg gaggctcgag atggtgagag 60 tgtaatcggt accagac 77 87381 DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence coding for version “b” of humanized L chain V region87 atg agg gcc cct gct cag ttt ttt ggg atc ttg ttg ctc tgg ttt cca 48Met Arg Ala Pro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15-10 -5 ggg atc cga tgt gac atc cag atg acc cag tct cca tcc tcc ctg tct96 Gly Ile Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 15 10 gca tct gta gga gac aga gtc acc atc acg tgc aag gcc tca cag gac 144Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25att aaa agc ttc tta agt tgg tat cag cag aaa cca ggg aaa gcc cct 192 IleLys Ser Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 30 35 40 aagctc ctg atc tat tat gca act agt ttg gca gat ggg gtc cca tca 240 Lys LeuLeu Ile Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 aggttc agt ggc tcc gga tct ggt acc gat tac act ctc acc atc tcg 288 Arg PheSer Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 65 70 75 agc ctccag cct gaa gat ttt gca act tac tat tgt ctg cag cat ggt 336 Ser Leu GlnPro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly 80 85 90 gag agc ccgtac acg ttc ggc gga ggg acc aag gtg gag atc aaa 381 Glu Ser Pro Tyr ThrPhe Gly Gly Gly Thr Lys Val Glu Ile Lys 95 100 105 88 107 PRT ArtificialSequence Description of Artificial Sequence Synthetic amino acidsequence of version “b” of humanized L chain V region 88 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 20 25 30 Leu Ser TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr AlaThr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly SerGly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu AspPhe Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Tyr 85 90 95 Thr PheGly Gly Gly Thr Lys Val Glu Ile Lys 100 105 89 381 DNA ArtificialSequence Description of Artificial Sequence Synthetic nucleotidesequence coding for version “c” of humanized L chain V region 89 atg agggcc cct gct cag ttt ttt ggg atc ttg ttg ctc tgg ttt cca 48 Met Arg AlaPro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 gggatc cga tgt gac atc cag atg acc cag tct cca tcc tcc ctg tct 96 Gly IleArg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 1 5 10 gcatct gta gga gac aga gtc acc atc acg tgc aag gcc tca cag gac 144 Ala SerVal Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 att aaaagc ttc tta agt tgg tat cag cag aaa cca ggg aaa gcc cct 192 Ile Lys SerPhe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 30 35 40 aag ctc ctgatc tat tat gca act agt ttg gca gat ggg gtc cca tca 240 Lys Leu Leu IleTyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 agg ttc agtggc tcc gga tct ggt acc gat tac act ctc acc atc tcg 288 Arg Phe Ser GlySer Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 65 70 75 agc ctc cag cctgaa gat att gca act tac tat tgt ctg cag cat ggt 336 Ser Leu Gln Pro GluAsp Ile Ala Thr Tyr Tyr Cys Leu Gln His Gly 80 85 90 gag agc ccg tac acgttc ggc gga ggg acc aag gtg gag atc aaa 381 Glu Ser Pro Tyr Thr Phe GlyGly Gly Thr Lys Val Glu Ile Lys 95 100 105 90 107 PRT ArtificialSequence Description of Artificial Sequence Synthetic amino acidsequence of version “c” of humanized L chain V region 90 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 20 25 30 Leu Ser TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr AlaThr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly SerGly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu AspIle Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Tyr 85 90 95 Thr PheGly Gly Gly Thr Lys Val Glu Ile Lys 100 105 91 72 DNA ArtificialSequence Description of Artificial Sequence Synthetic FR Shufflingprimer F2SS 91 gtctcttaag ttggttccag cagaaaccag ggaaatctcc taagaccctgatctactatg 60 caactagtaa ca 72 92 72 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic FR Shuffling primer F2SA 92 tgttactagttgcatagtag atcagggtct taggagattt ccctggtttc tgctggaacc 60 aacttaagag ac72 93 72 DNA Artificial Sequence Description of Artificial SequenceSynthetic FR Shuffling primer F2XS 93 gtctcttaag ttggtatcag cagaaaccagagaaagcccc taagtccctg atctattatg 60 caactagtaa ca 72 94 72 DNAArtificial Sequence Description of Artificial Sequence Synthetic FRShuffling primer F2XA 94 tgttactagt tgcataatag atcagggact taggggctttctctggtttc tgctgatacc 60 aacttaagag ac 72 95 381 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide sequence codingfor version “b1” of humanized L chain V region 95 atg agg gcc cct gctcag ttt ttt ggg atc ttg ttg ctc tgg ttt cca 48 Met Arg Ala Pro Ala GlnPhe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 ggg atc cga tgtgac atc cag atg acc cag tct cca tcc tcc ctg tct 96 Gly Ile Arg Cys AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 1 5 10 gca tct gta ggagac aga gtc acc atc acg tgc aag gcc tca cag gac 144 Ala Ser Val Gly AspArg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 att aaa agc ttc ttaagt tgg ttc cag cag aaa cca ggg aaa tct cct 192 Ile Lys Ser Phe Leu SerTrp Phe Gln Gln Lys Pro Gly Lys Ser Pro 30 35 40 aag acc ctg atc tac tatgca act agt ttg gca gat ggg gtc cca tca 240 Lys Thr Leu Ile Tyr Tyr AlaThr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 agg ttc agt ggc tcc ggatct ggt acc gat tac act ctc acc atc tcg 288 Arg Phe Ser Gly Ser Gly SerGly Thr Asp Tyr Thr Leu Thr Ile Ser 65 70 75 agc ctc cag cct gaa gat tttgca act tac tat tgt ctg cag cat ggt 336 Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Leu Gln His Gly 80 85 90 gag agc ccg tac acg ttc ggc ggaggg acc aag gtg gag atc aaa 381 Glu Ser Pro Tyr Thr Phe Gly Gly Gly ThrLys Val Glu Ile Lys 95 100 105 96 107 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “b1” of humanized L chain V region 96 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 20 25 30 Leu Ser Trp Phe GlnGln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Tyr Ala Thr SerLeu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly ThrAsp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe AlaThr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Tyr 85 90 95 Thr Phe Gly GlyGly Thr Lys Val Glu Ile Lys 100 105 97 381 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide sequence codingfor version “b2” of humanized L chain V region 97 atg agg gcc cct gctcag ttt ttt ggg atc ttg ttg ctc tgg ttt cca 48 Met Arg Ala Pro Ala GlnPhe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 ggg atc cga tgtgac atc cag atg acc cag tct cca tcc tcc ctg tct 96 Gly Ile Arg Cys AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 1 5 10 gca tct gta ggagac aga gtc acc atc acg tgc aag gcc tca cag gac 144 Ala Ser Val Gly AspArg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 att aaa agc ttc ttaagt tgg tat cag cag aaa cca gag aaa gcc cct 192 Ile Lys Ser Phe Leu SerTrp Tyr Gln Gln Lys Pro Glu Lys Ala Pro 30 35 40 aag tcc ctg atc tat tatgca act agt ttg gca gat ggg gtc cca tca 240 Lys Ser Leu Ile Tyr Tyr AlaThr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 agg ttc agt ggc tcc ggatct ggt acc gat tac act ctc acc atc tcg 288 Arg Phe Ser Gly Ser Gly SerGly Thr Asp Tyr Thr Leu Thr Ile Ser 65 70 75 agc ctc cag cct gaa gat tttgca act tac tat tgt ctg cag cat ggt 336 Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Leu Gln His Gly 80 85 90 gag agc ccg tac acg ttc ggc ggaggg acc aag gtg gag atc aaa 381 Glu Ser Pro Tyr Thr Phe Gly Gly Gly ThrLys Val Glu Ile Lys 95 100 105 98 107 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence ofversion “b2” of humanized L chain V region 98 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 20 25 30 Leu Ser Trp Tyr GlnGln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Tyr Ala Thr SerLeu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly ThrAsp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe AlaThr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Tyr 85 90 95 Thr Phe Gly GlyGly Thr Lys Val Glu Ile Lys 100 105 99 117 PRT Mus musculus Amino acidsequence of H chain V region of anti TF mouse monoclonal antibody ATR-599 Glu Val Gln Leu Gln Gln Ser Gly Thr Asn Leu Val Arg Pro Gly Ala 1 510 15 Leu Val Lys Leu Ser Cys Lys Gly Ser Gly Phe Asn Ile Lys Asp Tyr 2025 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 3540 45 Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp Pro Lys Phe 5055 60 Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 6570 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys85 90 95 Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser100 105 110 Val Thr Val Ser Ser 115 100 107 PRT Mus musculus Amino acidsequence of L chain V region of anti TF mouse monoclonal antibody ATR-5100 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 510 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 2025 30 Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro Lys Thr Leu Ile 3540 45 Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 5055 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Asn Asn Leu Glu Ser 6570 75 80 Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Tyr85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 101 780 DNAHomo sapiens DNA coding for soluble human TF 101 atg gag acc cct gcc tggccc cgg gtc ccg cgc ccc gag acc gcc gtc 48 Met Glu Thr Pro Ala Trp ProArg Val Pro Arg Pro Glu Thr Ala Val -30 -25 -20 gct cgg acg ctc ctg ctcggc tgg gtc ttc gcc cag gtg gcc ggc gct 96 Ala Arg Thr Leu Leu Leu GlyTrp Val Phe Ala Gln Val Ala Gly Ala -15 -10 -5 -1 tca ggc act aca aatact gtg gca gca tat aat tta act tgg aaa tca 144 Ser Gly Thr Thr Asn ThrVal Ala Ala Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 act aat ttc aag acaatt ttg gag tgg gaa ccc aaa ccc gtc aat caa 192 Thr Asn Phe Lys Thr IleLeu Glu Trp Glu Pro Lys Pro Val Asn Gln 20 25 30 gtc tac act gtt caa ataagc act aag tca gga gat tgg aaa agc aaa 240 Val Tyr Thr Val Gln Ile SerThr Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 tgc ttt tac aca aca gac acagag tgt gac ctc acc gac gag att gtg 288 Cys Phe Tyr Thr Thr Asp Thr GluCys Asp Leu Thr Asp Glu Ile Val 50 55 60 aag gat gtg aag cag acg tac ttggca cgg gtc ttc tcc tac ccg gca 336 Lys Asp Val Lys Gln Thr Tyr Leu AlaArg Val Phe Ser Tyr Pro Ala 65 70 75 80 ggg aat gtg gag agc acc ggt tctgct ggg gag cct ctg tat gag aac 384 Gly Asn Val Glu Ser Thr Gly Ser AlaGly Glu Pro Leu Tyr Glu Asn 85 90 95 tcc cca gag ttc aca cct tac ctg gagaca aac ctc gga cag cca aca 432 Ser Pro Glu Phe Thr Pro Tyr Leu Glu ThrAsn Leu Gly Gln Pro Thr 100 105 110 att cag agt ttt gaa cag gtg gga acaaaa gtg aat gtg acc gta gaa 480 Ile Gln Ser Phe Glu Gln Val Gly Thr LysVal Asn Val Thr Val Glu 115 120 125 gat gaa cgg act tta gtc aga agg aacaac act ttc cta agc ctc cgg 528 Asp Glu Arg Thr Leu Val Arg Arg Asn AsnThr Phe Leu Ser Leu Arg 130 135 140 gat gtt ttt ggc aag gac tta att tataca ctt tat tat tgg aaa tct 576 Asp Val Phe Gly Lys Asp Leu Ile Tyr ThrLeu Tyr Tyr Trp Lys Ser 145 150 155 160 tca agt tca gga aag aaa aca gccaaa aca aac act aat gag ttt ttg 624 Ser Ser Ser Gly Lys Lys Thr Ala LysThr Asn Thr Asn Glu Phe Leu 165 170 175 att gat gtg gat aaa gga gaa aactac tgt ttc agt gtt caa gca gtg 672 Ile Asp Val Asp Lys Gly Glu Asn TyrCys Phe Ser Val Gln Ala Val 180 185 190 att ccc tcc cga aca gtt aac cggaag agt aca gac agc ccg gta gag 720 Ile Pro Ser Arg Thr Val Asn Arg LysSer Thr Asp Ser Pro Val Glu 195 200 205 tgt atg ggc cag gag aaa ggg gaattc aga gaa gac tac aaa gac gat 768 Cys Met Gly Gln Glu Lys Gly Glu PheArg Glu Asp Tyr Lys Asp Asp 210 215 220 gac gat aaa taa 780 Asp Asp Lys225 102 259 PRT Homo sapiens Amino acid sequence of soluble human TF 102Met Glu Thr Pro Ala Trp Pro Arg Val Pro Arg Pro Glu Thr Ala Val -30 -25-20 Ala Arg Thr Leu Leu Leu Gly Trp Val Phe Ala Gln Val Ala Gly Ala -15-10 -5 -1 Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp LysSer 1 5 10 15 Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro ValAsn Gln 20 25 30 Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp LysSer Lys 35 40 45 Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp GluIle Val 50 55 60 Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser TyrPro Ala 65 70 75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro LeuTyr Glu Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu GlyGln Pro Thr 100 105 110 Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val AsnVal Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn ThrPhe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu Ile Tyr ThrLeu Tyr Tyr Trp Lys Ser 145 150 155 160 Ser Ser Ser Gly Lys Lys Thr AlaLys Thr Asn Thr Asn Glu Phe Leu 165 170 175 Ile Asp Val Asp Lys Gly GluAsn Tyr Cys Phe Ser Val Gln Ala Val 180 185 190 Ile Pro Ser Arg Thr ValAsn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Met Gly Gln GluLys Gly Glu Phe Arg Glu Asp Tyr Lys Asp Asp 210 215 220 Asp Asp Lys 225103 136 PRT Mus musculus 103 Met Lys Cys Ser Trp Val Ile Phe Phe Leu MetAla Val Val Thr Gly -15 -10 -5 Val Asn Ser Glu Val Gln Leu Gln Gln SerGly Thr Asn Leu Val Arg -1 1 5 10 Pro Gly Ala Leu Val Lys Leu Ser CysLys Gly Ser Gly Phe Asn Ile 15 20 25 Lys Asp Tyr Tyr Met His Trp Val LysGln Arg Pro Glu Gln Gly Leu 30 35 40 45 Glu Trp Ile Gly Gly Asn Asp ProAla Asn Gly His Ser Met Tyr Asp 50 55 60 Pro Lys Phe Gln Gly Lys Ala SerIle Thr Ala Asp Thr Ser Ser Asn 65 70 75 Thr Ala Tyr Leu Gln Leu Ser SerLeu Thr Ser Glu Asp Thr Ala Val 80 85 90 Tyr Phe Cys Ala Arg Asp Ser GlyTyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 Gly Thr Ser Val Thr Val SerSer 110 115 104 127 PRT Mus musculus 104 Met Arg Ala Pro Ala Gln Phe PheGly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 Gly Ile Arg Cys Asp IleLys Met Thr Gln Ser Pro Ser Ser Met Tyr -1 1 5 10 Ala Ser Leu Gly GluArg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 Ile Lys Ser Phe LeuSer Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro 30 35 40 Lys Thr Leu Ile TyrTyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 Arg Phe Ser GlySer Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Asn 65 70 75 Asn Leu Glu SerAsp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly 80 85 90 Glu Ser Pro TyrThr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 95 100 105 105 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “a” of humanized H chain V region 105 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Ala Lys Leu Thr Ala Ala Thr Ser Ala Ser 65 7075 Ile Ala Tyr Leu Glu Phe Ser Ser Leu Thr Asn Glu Asp Ser Ala Val 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 106 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “b” of humanized H chain V region 106 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn 65 7075 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 107 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “c” of humanized H chain V region 107 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn 65 7075 Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 108 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “d” of humanized H chain V region 108 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 65 7075 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val 80 8590 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 109 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “e” of humanized H chain V region 109 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Ser Ile Thr Ala Asp Glu Ser Thr Lys 65 7075 Ile Ala Tyr Met Glu Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Val 80 8590 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 110 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “f” of humanized H chain V region 110 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 65 7075 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 111 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “g” of humanized H chain V region 111 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser 65 7075 Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val 80 8590 Tyr Ser Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 112 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “h” of humanized H chain 112 Met Lys Cys SerTrp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 Val Asn SerGln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10 Pro GlyThr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25 Lys AspTyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 GluTrp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 55 60 ProLys Phe Gln Gly Arg Val Thr Met Ser Ala Asp Lys Ser Ser Ser 65 70 75 AlaAla Tyr Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile 80 85 90 TyrPhe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 113 138 PRT ArtificialSequence Description of Artificial Sequence Synthetic amino acidsequence of version “i” of humanized H chain V region 113 Met Lys CysSer Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 Val AsnSer Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10 ProGly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25 LysAsp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 55 60Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 65 70 75Thr Val Phe Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95 100105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 114 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “j” of humanized H chain V region 114 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Phe Thr Ala Asp Thr Ser Ala Asn 65 7075 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Ala Asp Thr Ala Val 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 115 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “b1” of humanized H chain V region 115 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Met Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn 65 7075 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 116 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “d1” of humanized H chain V region 116 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Met Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 65 7075 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val 80 8590 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 117 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “b3” of humanized H chain V region 117 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn 65 7075 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile 80 8590 Tyr Tyr Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 118 138 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “d3” of humanized H chain V region 118 Met LysCys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly -15 -10 -5 ValAsn Ser Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg -1 1 5 10Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 4045 Glu Trp Ile Gly Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 50 5560 Pro Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 65 7075 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val 80 8590 Tyr Phe Cys Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln 95100 105 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 110 115 119 127 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence of version “a” of humanized L chain V region 119 Met ArgAla Pro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5Gly Ile Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 1 510 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 2025 Ile Lys Ser Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 30 3540 Lys Leu Leu Ile Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser 45 5055 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 6570 75 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly 8085 90 Glu Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 95 100105 120 127 PRT Artificial Sequence Description of Artificial SequenceSynthetic amino acid sequence of version “b” of humanized L chain Vregion 120 Met Arg Ala Pro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp PhePro -20 -15 -10 -5 Gly Ile Arg Cys Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser -1 1 5 10 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asp 15 20 25 Ile Lys Ser Phe Leu Ser Trp Tyr Gln Gln Lys ProGly Lys Ala Pro 30 35 40 Lys Leu Leu Ile Tyr Tyr Ala Thr Ser Leu Ala AspGly Val Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp TyrThr Leu Thr Ile Ser 65 70 75 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr TyrCys Leu Gln His Gly 80 85 90 Glu Ser Pro Tyr Thr Phe Gly Gly Gly Thr LysVal Glu Ile Lys 95 100 105 121 127 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic amino acid sequence of version “c” ofhumanized L chain V region 121 Met Arg Ala Pro Ala Gln Phe Phe Gly IleLeu Leu Leu Trp Phe Pro -20 -15 -10 -5 Gly Ile Arg Cys Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser -1 1 5 10 Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 Ile Lys Ser Phe Leu Ser TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro 30 35 40 Lys Leu Leu Ile Tyr Tyr AlaThr Ser Leu Ala Asp Gly Val Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser GlySer Gly Thr Asp Tyr Thr Leu Thr Ile Ser 65 70 75 Ser Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Leu Gln His Gly 80 85 90 Glu Ser Pro Tyr Thr PheGly Gly Gly Thr Lys Val Glu Ile Lys 95 100 105 122 127 PRT ArtificialSequence Description of Artificial Sequence Synthetic amino acidsequence of version “b1” of humanized L chain V region 122 Met Arg AlaPro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp Phe Pro -20 -15 -10 -5 GlyIle Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser -1 1 5 10Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25Ile Lys Ser Phe Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro 30 35 40Lys Thr Leu Ile Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser 45 50 5560 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 65 7075 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Gly 80 8590 Glu Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 95 100105 123 127 PRT Artificial Sequence Description of Artificial SequenceSynthetic amino acid sequence of version “b2” of humanized L chain Vregion 123 Met Arg Ala Pro Ala Gln Phe Phe Gly Ile Leu Leu Leu Trp PhePro -20 -15 -10 -5 Gly Ile Arg Cys Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser -1 1 5 10 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asp 15 20 25 Ile Lys Ser Phe Leu Ser Trp Tyr Gln Gln Lys ProGlu Lys Ala Pro 30 35 40 Lys Ser Leu Ile Tyr Tyr Ala Thr Ser Leu Ala AspGly Val Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp TyrThr Leu Thr Ile Ser 65 70 75 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr TyrCys Leu Gln His Gly 80 85 90 Glu Ser Pro Tyr Thr Phe Gly Gly Gly Thr LysVal Glu Ile Lys 95 100 105

1. A blood rheology-improving agent comprising an antibody against humantissue factor (human TF).
 2. The blood rheology-improving agentaccording to claim 1 wherein said antibody is a polyclonal antibody. 3.The blood rheology-improving agent according to claim 1 wherein saidantibody is a monoclonal antibody.
 4. The blood rheology-improving agentaccording to claim 1 or 3 wherein said antibody is a recombinantantibody.
 5. The blood rheology-improving agent according to claim 1 or4 wherein said antibody is an altered antibody.
 6. The bloodrheology-improving agent according to claim 1, 4 or 5 wherein saidaltered antibody is a chimeric antibody or a humanized antibody.
 7. Theblood rheology-improving agent according to claim 6 wherein saidhumanized antibody is a humanized antibody of version b-b, i-b, or i-b2.8. The blood rheology-improving agent according to any one of claims 1,or 4-7 wherein said antibody is an antibody modification.
 9. The bloodrheology-improving agent according to claim 8 wherein said antibodymodification is an antibody fragment Fab, F(ab′)₂, or Fv, or a singlechain Fv (scFv).
 10. The use of an antibody against human tissue factor(human TF) for the production of a blood rheology-improving agent. 11.The use according to claim 10 wherein said antibody is a polyclonalantibody.
 12. The use according to claim 10 wherein said antibody is amonoclonal antibody.
 13. The use according to claim 10 or 12 whereinsaid antibody is a recombinant antibody.
 14. The use according to claim10 or 13 wherein said antibody is an altered antibody.
 15. The useaccording to claim 10, 13 or 14 wherein said altered antibody is achimeric antibody or a humanized antibody.
 16. The use according toclaim 15 wherein said humanized antibody is a humanized antibody ofversion b-b, i-b, or i-b2.
 17. The use according to any one of claims10, or 13-16 wherein said antibody is an antibody modification.
 18. Theuse according to claim 17 wherein said antibody modification is anantibody fragment Fab, F(ab′)₂, or Fv, or a single chain Fv (scFv). 19.A method of improving blood rheology comprising administering anantibody against human tissue factor (human TF).
 20. The methodaccording to claim 19 wherein said antibody is a polyclonal antibody.21. The method according to claim 19 wherein said antibody is amonoclonal antibody.
 22. The method according to claim 19 or 21 whereinsaid-antibody is a recombinant antibody.
 23. The method according toclaim 19 or 22 wherein said antibody is an altered antibody.
 24. Themethod according to claim 19, 22 or 23 wherein said altered antibody isa chimeric antibody or a humanized antibody.
 25. The method according toclaim 24 wherein said humanized antibody is a humanized antibody ofversion b-b, i-b, or i-b2.
 26. The method according to any one of claims19, or 22-25 wherein said antibody is an antibody modification.
 27. Themethod according to claim 26 wherein said antibody modification is anantibody fragment Fab, F(ab′)₂, or Fv, or a single chain Fv (scFv).